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Patent 2488404 Summary

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(12) Patent: (11) CA 2488404
(54) English Title: GENES AND POLYPEPTIDES RELATING TO HUMAN COLON CANCERS
(54) French Title: GENES ET POLYPEPTIDES EN RAPPORT AVEC LES CANCERS DU COLON CHEZ L'HOMME
Status: Expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/11 (2006.01)
  • A61K 31/713 (2006.01)
  • A61K 38/00 (2006.01)
  • A61K 39/00 (2006.01)
  • C07K 14/47 (2006.01)
  • C07K 16/32 (2006.01)
  • C12N 15/12 (2006.01)
  • G01N 33/68 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • NAKAMURA, YUSUKE (Japan)
  • FURUKAWA, YOICHI (Japan)
  • TAHARA, HIDEAKI (Japan)
  • TSUNODA, TAKUYA (Japan)
(73) Owners :
  • ONCOTHERAPY SCIENCE, INC. (Japan)
(71) Applicants :
  • ONCOTHERAPY SCIENCE, INC. (Japan)
  • THE UNIVERSITY OF TOKYO (Japan)
(74) Agent: BERESKIN & PARR LLP/S.E.N.C.R.L.,S.R.L.
(74) Associate agent:
(45) Issued: 2012-11-27
(86) PCT Filing Date: 2003-06-03
(87) Open to Public Inspection: 2003-12-18
Examination requested: 2008-06-02
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/JP2003/007006
(87) International Publication Number: WO2003/104275
(85) National Entry: 2004-12-03

(30) Application Priority Data:
Application No. Country/Territory Date
60/386,985 United States of America 2002-06-06
60/415,209 United States of America 2002-09-30
60/451,013 United States of America 2003-02-28

Abstracts

English Abstract




The present application provides novel human genes RNF43 whose expression is
markedly elevated in colorectal cancers, as well as CXADRL1 and GCUD1 whose
expression is markedly elevated in gastric cancers compared to corresponding
non-cancerous tissues. The genes and polypeptides encoded by the genes can be
used, for example, in the diagnosis of a cell proliferative disease, and as
target molecules for developing drugs against the disease.


French Abstract

La présente invention concerne les nouveaux gènes humains RNF43 dont l'expression est nettement élevée dans les cancers colorectaux, ainsi que CXADRL1 et GCUD1 dont l'expression est nettement élevée dans les cancers de l'estomac comparativement aux tissus non cancéreux correspondants. Les gènes et les polypeptides codés par les gènes peuvent être utilisés, par exemple, dans le diagnostic d'une maladie proliférative cellulaire et en tant que molécules cibles utiles pour mettre au point des médicaments s'opposant à la maladie.

Claims

Note: Claims are shown in the official language in which they were submitted.



143
CLAIMS

1. A substantially pure polypeptide comprising the amino acid sequence of SEQ
ID NO: 6.
2. An isolated polynucleotide encoding the polypeptide of claim 1,

3. A vector comprising the polynucleotide of claim 2.

4. An isolated host cell harboring the polynucleotide of claim 2 or the vector
of claim 3.

5. A method for producing the polypeptide of claim 1, said method comprising
the steps of:
(a) culturing the host cell of claim 4;

(b) allowing the host cell to express the polypeptide; and
(c) collecting the expressed polypeptide.

6. A polynucleotide that is the complementary strand of the polynucleotide of
claim 2.

7. An antisense polynucleotide against the polynucleotide of claim 2,
comprising the
nucleotide sequence of SEQ ID NO: 31.

8. A small interfering RNA against the polynucleotide of claim 2, wherein the
sense strand
thereof comprises the nucleotide sequence of SEQ ID NO: 112 or 113, as the
target
sequence.

9. A method for diagnosing colon cancer, said method comprising the steps of:

(a) detecting the expression level of the gene encoding the amino acid
sequence of
SEQ ID NO: 6 in a biological sample of specimen; and

(b) diagnosing a subject as being affected with colon cancer, when an
elevation of the
expression level compared with that in a normal sample is detected in step
(a).

10. The method of claim 9, wherein the expression level is detected by a
method selected
from the group consisting of:

(a) detecting the mRNA encoding the amino acid sequence of SEQ ID NO: 6;

(b) detecting the protein comprising the amino acid sequence of SEQ ID NO: 6;
and


144
(c) detecting the biological activity to promote cell proliferation of the
protein

comprising the amino acid sequence of SEQ ID NO: 6.

11. An in vitro method of screening for a compound for treating colon cancer,
said method
comprising the steps of:

(a) contacting a candidate compound with a cell expressing a polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 5; and

(b) selecting a compound that reduces the expression level of the
polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 5 in comparison with the
expression level detected in the absence of the test compound.

12. An in vitro method of screening for a compound for treating colon cancer,
said method
comprising the steps of:

(a) contacting a test compound with a polypeptide selected from the group
consisting
of:

(i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; and
(ii) a polypeptide encoded by a polynucleotide that hybridizes under stringent

conditions to the complement of a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO: 5, wherein the polypeptide has a
biological activity to promote cell proliferation equivalent to a polypeptide
consisting of the amino acid sequence of SEQ ID NO: 6, wherein the
stringent condition is defined with washing 3 times in 2X SSC, 0.01%
SDS at room temperature for 20 min, then washing 3 times in 1X SSC,
0.1% SDS at 37°C for 20 min, and washing twice in 1X SSC, 0.1% SDS
at 50°C for 20 min;


145
(b) detecting the biological activity to promote cell proliferation of the
polypeptide of
step (a); and

(c) selecting a compound that suppresses the biological activity of the
polypeptide in
comparison with the biological activity detected in the absence of the test
compound.

13. A method of screening for a compound for treating colon cancer, said
method comprising
the steps of:

(a) contacting a candidate compound with a cell into which a vector comprising
the
transcriptional regulatory region of a marker gene consisting of SEQ ID NO: 5
and a reporter gene that is expressed under the control of the transcriptional
regulatory region has been introduced;

(b) measuring the activity of said reporter gene; and

(c) selecting a compound that reduces the expression level of said reporter
gene as
compared to a control.

14. A composition for treating colon cancer, said composition comprising a
pharmaceutically
effective amount of an antisense polynucleotide comprising the nucleotide
sequence of
SEQ ID NO: 31 or small interfering RNA comprising the nucleotide sequence of
SEQ ID
NO: 112 as the target sequence, and a pharmaceutically acceptable carrier.

15. Use of an antisense polynucleotide comprising the nucleotide sequence of
SEQ ID NO:
31 or small interfering RNA comprising the nucleotide sequence of SEQ ID NO:
112 as
the target sequence for treating colon cancer.

16. Use of a polypeptide selected from the group consisting of (a)-(c), or a
polynucleotide
encoding the polypeptide:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6;


146
(b) a polypeptide encoded by a polynucleotide that hybridizes under stringent

conditions to the complement of a polynucleotide consisting of the nucleotide
sequence of SEQ ID NO: 5, wherein the polypeptide has a biological activity to

promote cell proliferation equivalent to a polypeptide consisting of the amino
acid
sequence of SEQ ID NO: 6, wherein the stringent condition is defined with
washing 3 times in 2X SSC, 0.01% SDS at room temperature for 20 min, then
washing 3 times in 1X SSC, 0.1% SDS at 37°C for 20 min, and washing
twice in
1X SSC, 0.1% SDS at 50°C for 20 min; and

(c) a polypeptide selected from the group of polypeptides comprising the amino
acid
sequence of SEQ ID NO: 80, 97, and 108;

for treating or preventing colon cancer.

17. Use of a polypeptide selected from the group consisting of (a)-(c), a
polynucleotide
encoding the polypeptide or a vector comprising the polynucleotide:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under stringent
conditions to the complement of a polynucleotide consisting of the nucleotide
sequence of SEQ ID NO: 5, wherein the polypeptide has a biological activity to

promote cell proliferation equivalent to a polypeptide consisting of the amino
acid
sequence of SEQ ID NO: 6, wherein the stringent condition is defined with
washing 3 times in 2X SSC, 0.01% SDS at room temperature for 20 min, then
washing 3 times in 1X SSC, 0.1% SDS at 37°C for 20 min, and washing
twice in
1X SSC, 0.1% SDS at 50°C for 20 min; and

(c) a polypeptide selected from the group of polypeptides comprising the amino
acid
sequence of SEQ ID NO: 80, 97, and 108;


147
for inducing an anti tumor immunity for colon cancer.

18. The use according to claim 17, wherein the use further comprises the use
of antigen
presenting cells.

19. A pharmaceutical composition for treating or preventing a colon cancer,
said composition
comprising a pharmaceutically acceptable carrier and a pharmaceutically
effective
amount of at least one polypeptide selected from the group of (a)-(b), or a
polynucleotide
encoding the polypeptide:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; and

(b) a polypeptide consisting of the amino acid sequence selected from the
group of
SEQ ID NO: 80, 97, and 108.

20. The pharmaceutical composition of claim 19, wherein the polynucleotide is
incorporated
in an expression vector.

21. A polypeptide consisting of the amino acid sequence selected from the
group of SEQ ID
NO: 80, 97, and 108.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02488404 2010-12-21

WO 03/104275 PCT/JP03/07006
1

DESCRIPTION
GENES AND POLYPEPTIDES RELATING TO HUMAN COLON CANCERS
Technical Field
The present invention relates to the field of biological science, more
specifically to
the field of cancer research. In particular, the present invention relates to
novel genes,
RNF43, CXADRL1, and GCUD1, involved in the proliferation mechanism of cells,
as well
as polypeptides encoded by the genes. The genes and polypeptides of the
present
invention can be used, for example, in the diagnosis of cell proliferative
disease, and as
target molecules for developing drugs against the disease.
Background Art
Gastric cancers and colorectal cancers are leading causes of cancer death
worldwide. In spite of recent progress in diagnostic and therapeutic
strategies, prognosis
of patients with advanced cancers remains very poor. Although molecular
studies have
revealed the involvement of alterations in tumor suppressor genes and/or
oncogenes in
carcinogenesis, the precise mechanisms still remain to be elucidated.
cDNA microarray technologies have enabled to obtain comprehensive profiles of
gene expression in normal and malignant cells, and compare the gene expression
in
malignant and corresponding normal cells (Okabe et al., Cancer Res 61:2129-37
(2001);
Kitahara et al., Cancer Res 61: 3544-9 (2001); Lin et al., Oncogene 21:4120-8
(2002);
Hasegawa et al., Cancer Res 62:7012-7 (2002)). This approach enables to
disclose the
complex nature of cancer cells, and helps to understand the mechanism of
carcinogenesis.
Identification of genes that are deregulated in tumors can lead to more
precise and accurate
diagnosis of individual cancers, and to develop novel therapeutic targets
(Bienz and
Clevers, Cell 103:311-20 (2000)). To disclose mechanisms underlying tumors
from a
genome-wide point of view, and discover target molecules for diagnosis and
development
of novel therapeutic drugs, the present inventors have been analyzing the
expression
profiles of tumor cells using a cDNA microarray of 23040 genes (Okabe et al.,
Cancer Res
61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al.,
Oncogene
21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).
Studies designed to reveal mechanisms of carcinogenesis have already
facilitated


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2

identification of molecular targets for anti-tumor agents. For example,
inhibitors of
farnexyltransferase (FTIs) which were originally developed to inhibit the
growth-signaling
pathway related to Ras, whose activation depends on posttranslational
farnesylation, has
been effective in treating Ras-dependent tumors in animal models (He et al.,
Cell
99:335-45 (1999)). Clinical trials on human using a combination or anti-cancer
drugs and
anti-HER2 monoclonal antibody, trastuzumab, have been conducted to antagonize
the
proto-oncogene receptor HER2/neu; and have been achieving improved clinical
response
and overall survival of breast-cancer patients (Lin et al., Cancer Res 61:6345-
9 (2001)).
A tyrosine kinase inhibitor, STI-571, which selectively inactivates bcr-abl
fusion proteins,
has been developed to treat chronic myelogenous leukemias wherein constitutive
activation
of bcr-abl tyrosine kinase plays a crucial role in the transformation of
leukocytes. Agents
of these kinds are designed to suppress oncogenic activity of specific gene
products (Fujita
et al., Cancer Res 61:7722-6 (2001)). Therefore, gene products commonly up-
regulated
in cancerous cells may serve as potential targets for developing novel anti-
cancer agents.
It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs) recognize
epitope peptides derived from tumor-associated antigens (TAAs) presented on
MHC Class
I molecule, and lyse tumor cells. Since the discovery of MAGE family as the
first
example of TAAs, many other TAAs have been discovered using immunological
approaches (Boon, Int J Cancer 54: 177-80 (1993); Boon and van der Bruggen, J
Exp Med
183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991);
Brichard et al., J
Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994)).
Some of
the discovered TAAs are now in the stage of clinical development as targets of
immunotherapy. TAAs discovered so far include MAGE (van der Bruggen et al.,
Science
254: 1643-7 (1991)), gp100 (Kawakami et al., J Exp Med 180: 347-52 (1994)),
SART
(Shichijo et al., J Exp Med 187: 277-88 (1998)), and NY-ESO-1 (Chen et al.,
Proc Natl
Acad Sci USA 94: 1914-8 (1997)). On the other hand, gene products which had
been
demonstrated to be specifically overexpressed in tumor cells, have been shown
to be
recognized as targets inducing cellular immune responses. Such gene products
include
p53 (Umano et al., Brit J Cancer 84: 1052-7 (2001)), HER2/neu (Tanaka et al.,
Brit J
Cancer 84: 94-9 (2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)),
and so on.
In spite of significant progress in basic and clinical research concerning
TAAs
(Rosenbeg et al., Nature Med 4: 321-7 (1998); Mukherji et al., Proc Natl Acad
Sci USA 92:
8078-82 (1995); Hu et al., Cancer Res 56: 2479-83 (1996)), only limited number
of
candidate TAAs for the treatment of adenocarcinomas, including colorectal
cancer, are
available. TAAs abundantly expressed in cancer cells, and at the same time
which
expression is restricted to cancer cells would be promising candidates as


CA 02488404 2004-12-03
WO 03/104275 PCT/JP03/07006
3

immunotherapeutic targets. Further, identification of new TAAs inducing potent
and
specific antitumor immune responses is expected to encourage clinical use of
peptide
vaccination strategy in various types of cancer (Boon and can der Bruggen, J
Exp Med
183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991);
Brichard et al., J
Exp Med 178: 489-95 (1993); Kawakami et al.,J Exp Med 180: 347-52 (1994);
Shichijo et
al., J Exp Med 187: 277-88 (1998); Chen et al., Proc Natl Acad Sci USA 94:
1914-8
(1997); Harris, J Natl Cancer Inst 88: 1442-5 (1996); Butterfield et al.,
Cancer Res 59:
3134-42 (1999); Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et
al., J
Immunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8 (1997);
Fujie et al.,
Int J Cancer 80: 169-72 (1999); Kikuchi et al., Int J Cancer 81: 459-66
(1999); Oiso et al.,
Int J Cancer 81: 387-94 (1999)).
It has been repeatedly reported that peptide-stimulated peripheral blood
mononuclear cells (PBMCs) from certain healthy donors produce significant
levels of
IFN-y in response to the peptide, but rarely exert cytotoxicity against tumor
cells in an
HLA-A24 or -A0201 restricted manner in 51Cr-release assays (Kawano et al.,
Cance Res
60: 3550-8 (2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et
al., Jpn J
Cancer Res 92: 762-7 (2001)). However, both of HLA-A24 and HLA-A0201 are one
of
the popular HLA alleles in Japanese, as well as Caucasian (Date et al., Tissue
Antigens 47:
93-101 (1996); Kondo et al., J Immunol 155: 4307-12 (1995); Kubo et al., J
Immunol 152:
3913-24 (1994); Imanishi et al., Proceeding of the eleventh International
Hictocompatibility Workshop and Conference Oxford University Press, Oxford,
1065
(1992); Williams et al., Tissue Antigen 49: 129 (1997)). Thus, antigenic
peptides of
cancers presented by these HLAs may be especially useful for the treatment of
cancers
among Japanese and Caucasian. Further, it is known that the induction of low-
affinity
CTL in vitro usually results from the use of peptide at a high concentration,
generating a
high level of specific peptide/MHC complexes on antigen presenting cells
(APCs), which
will effectively activate these CTL (Alexander-Miller et al., Proc Natl Acad
Sci USA 93:
4102-7 (1996)).

Summary of the Invention
An object of the present invention is to provide novel proteins involved in
the
proliferation mechanism of gastric or colorectal cancer cells and the genes
encoding the
proteins, as well as methods for producing and using the same in the diagnosis
and
treatment of gastric cancer or colorectal cancer.
To disclose the mechanism of gastric and colorectal carcinogenesis and
identify
novel diagnostic markers and/or drug targets for the treatment of these
tumors, the present


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4

inventors analyzed the expression profiles of genes in gastric and colorectal
carcinogenesis
using a genome-wide cDNA microarray containing 23040 genes. From the
pharmacological point of view, suppressing oncogenic signals is easier in
practice than
activating tumor-suppressive effects. Thus, the present inventors searched for
genes that
are up-regulated during gastric and colorectal carcinogenesis.
Among the transcripts that were commonly up-regulated in gastric cancers,
novel
human genes CXADRL1 (coxsackie and adenovirus receptor like 1) and GCUD1
(up-regulated in gastric cancer) were identified on chromosome band 3q13 and
7p14,
respectively. Gene transfer of CXADRLI or GCUD1 promoted proliferation of
cells.
Furthermore, reduction of CXADRL1 or GCUD1 expression by transfection of their
specific antisense S-oligonucleotides or small interfering RNAs inhibited the
growth of
gastric cancer cells. Many anticancer drugs, such as inhibitors of DNA and/or
RNA
synthesis, metabolic suppressors, and DNA intercalators, are not only toxic to
cancer cells
but also for normally growing cells. However, agents suppressing the
expression of
CXADRLI may not adversely affect other organs due to the fact that normal
expression of
the gene is restricted to the testis and ovary, and thus may be of great
importance for
treating cancer.
Further, among the transcripts that were commonly up-regulated in colorectal
cancers,
gene RNF43 (Ring finger protein 43) assigned at chromosomal band 17pter-p13.1
was
identified. In addition, yeast two-hybrid screening assay revealed that RNF43
protein
associated with NOTCH2 or STRIN.
NOTCH2 is a large transmembrane receptor protein that is a component of an
evolutionarily conserved intercellular signaling mechanism. NOTCH2 is a
protein
member of the Notch signaling pathway and is reported to be involved in
glomerulogenesis
in the kidney and development of heart and eye vasculature (McCright et al.,
Development
128: 491-502 (2001)). Three Delta/Serrate/Lag-2 (DSL) proteins, Deltal,
Jaggaedl, and
Jaggaed2, are reported as functional ligands for NOTCH2 (Shimizu et al., Mol
Cell Biol
20: 6913-22 (2000)). The signal induced by ligand binding in the Notch
signaling
pathway is transmitted intracellulaly by a process involving proteolysis of
the receptor and
nuclear translocation of the intracellular domain of the NOTCH protein (see
reviews
Artavanis-Tsakonas et al., Annu Rev Cell Biol 7: 427-52 (1999); Weinmaster,
Curr Opin
Genet Dev 10: 363-9 (2000)). Furthermore, reduction of RNF43 expression by
transfection of specific antisense S-oligonucleotides or small interfering
RNAs
corresponding to RNF43 inhibited the growth of colorectal cancer cells. As
already
described above many anticancer drugs, are not only toxic to cancer cells but
also for
normally growing cells. However, agents suppressing the expression of RNF43
may also


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not adversely affect other organs due to the fact that normal expression of
the gene is
restricted to fetus, more specifically fetal lung and fetal kidney, and thus
may be of great
importance for treating cancer.
Thus, the present invention provides isolated novel genes, CXADRL1, GCUD1, and
5 RNF43, which are candidates as diagnostic markers for cancer as well as
promising
potential targets for developing new strategies for diagnosis and effective
anti-cancer
agents. Further, the present invention provides polypeptides encoded by these
genes, as
well as the production and the use of the same. More specifically, the present
invention
provides the following:
The present application provides novel human polypeptides, CXADRL1, GCUD1,
and RNF43, or a functional equivalent thereof, that promotes cell
proliferation and is
up-regulated in cell proliferative diseases, such as gastric and colorectal
cancers.
In a preferred embodiment, the CXADRL1 polypeptide includes a putative 431
amino acid protein with about 37% identity to CXADR (coxsackie and adenovirus
receptor). CXADRL1 is encoded by the open reading frame of SEQ ID NO: 1 and
contains two immunogloblin domains at codons 29-124 and 158-232, as well as a
transmembrane domain at codons 246-268. The CXADRL1 polypeptide preferably
includes the amino acid sequence set forth in SEQ ID NO: 2. The present
application
also provides an isolated protein encoded from at least a portion of the
CXADRLI
polynucleotide sequence, or polynucleotide sequences at least 30%, and more
preferably at
least 40% complementary to the sequence set forth in SEQ ID NO: 1.
On the other hand, in a preferred embodiment, the GCUD1 polypeptide includes a
putative 414 amino acid protein encoded by the open reading frame of SEQ ID
NO: 3.
The GCUD1 polypeptide preferably includes the amino acid sequence set forth in
SEQ ID
NO: 4. The present application also provides an isolated protein encoded from
at least a
portion of the GCUD1 polynucleotide sequence, or polynucleotide sequences at
least 15%,
and more preferably at least 25% complementary to the sequence set forth in
SEQ ID NO:
3.
Furthermore, in a preferred embodiment, the RNF43 polypeptide includes a
putative 783 amino acid protein encoded by the open reading frame of SEQ ID
NO: 5.
The RNF43 polypeptide preferably includes the amino acid sequence set forth in
SEQ ID
NO: 6 and contains a Ring finger motif at codons 272-312. The RNF43
polypeptide
showed 38% homology to RING finger protein homolog DKFZp566HO73.1 (GenBank
Accession Number: T08729). The present application also provides an isolated
protein
encoded from at least a portion of the RNF43 polynucleotide sequence, or
polynucleotide
sequences at least 30%, and more preferably at least 40% complementary to the
sequence


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set forth in SEQ ID NO: 5.
The present invention further provides novel human genes, CXADRLI and GCUD1,
whose expressions are markedly elevated in a great majority of gastric cancers
as
compared to corresponding non-cancerous mucosae. In addition to gastric
cancers,
CXADRL1 and GCUD1 were also highly expressed in colorectal cancer and liver
cancer.
The isolated CXADRL1 gene includes a polynucleotide sequence as described in
SEQ ID
NO: 1. In particular, the CXADRLI cDNA includes 3423 nucleotides that contain
an open
reading frame of 1296 nucleotides (SEQ ID NO: 1). The present invention
further
encompasses polynucleotides which hybridize to and which are at least 30%, and
more
preferably at least 40% complementary to the polynucleotide sequence set forth
in SEQ ID
NO: 1, to the extent that they encode a CXADRL1 protein or a functional
equivalent
thereof. Examples of such polynucleotides are degenerates and allelic mutants
of SEQ ID
NO: 1. On the other hand, the isolated GCUD1 gene includes a polynucleotide
sequence
as described in SEQ ID NO: 3. In particular, the GCUD1 cDNA includes 4987
nucleotides that contain an open reading frame of 1245 nucleotides (SEQ ID NO:
3). The
present invention further encompasses polynucleotides which hybridize to and
which are at
least 15%, and more preferably at least 25% complementary to the
polynucleotide
sequence set forth in SEQ ID NO: 3, to the extent that they encode a GCUD1
protein or a
functional equivalent thereof. Examples of such polynucleotides are
degenerates and
allelic mutants of SEQ ID NO: 3.
Furthermore, the present invention provides a novel human gene RNF43, whose
expression is markedly elevated in a great majority of colorectal cancers as
compared to
corresponding non-cancerous mucosae. In addition to colorectal cancers, RNF43
was
also highly expressed in lung cancer, gastric cancer, and liver cancer. The
isolated
RNF43 gene includes a polynucleotide sequence as described in SEQ ID NO: 5. In
particular, the RNF43 cDNA includes 5345 nucleotides that contain an open
reading frame
of 2352 nucleotides (SEQ ID NO: 5). The present invention further encompasses
polynucleotides which hybridize to and which are at least 30%, and more
preferably at
least 40% complementary to the polynucleotide sequence set forth in SEQ ID NO:
5, to the
extent that they encode a RNF43 protein or a functional equivalent thereof.
Examples of
such polynucleotides are degenerates and allelic mutants of SEQ ID NO: 5.
As used herein, an isolated gene is a polynucleotide the structure of which is
not
identical to that of any naturally occurring polynucleotide or to that of any
fragment of a
naturally occurring genomic polynucleotide spanning more than three separate
genes.
The term therefore includes, for example, (a) a DNA which has the sequence of
part of a
naturally occurring genomic DNA molecule in the genome of the organism in
which it


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naturally occurs; (b) a polynucleotide incorporated into a vector or into the
genomic DNA
of a prokaryote or eukaryote in a manner such that the resulting molecule is
not identical to
any naturally occurring vector or genomic DNA; (c) a separate molecule such as
a cDNA,
a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or
a
restriction fragment; and (d) a recombinant nucleotide sequence that is part
of a hybrid
gene, i.e., a gene encoding a fusion polypeptide.
Accordingly, in one aspect, the invention provides an isolated polynucleotide
that
encodes a polypeptide described herein or a fragment thereof. Preferably, the
isolated
polypeptide includes a nucleotide sequence that is at least 60% identical to
the nucleotide
sequence shown in SEQ ID NO: 1, 3, or 5. More preferably, the isolated nucleic
acid
molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID
NO: 1, 3,
or 5. In the case of an isolated polynucleotide which is longer than or
equivalent in length
to the reference sequence, e.g., SEQ ID NO: 1, 3, or 5, the comparison is made
with the
full length of the reference sequence. Where the isolated polynucleotide is
shorter than
the reference sequence, e.g., shorter than SEQ ID NO: 1, 3, or 5, the
comparison is made to
segment of the reference sequence of the same length (excluding any loop
required by the
homology calculation).
The present invention also provides a method of producing a protein by
transfecting
or transforming a host cell with a polynucleotide sequence encoding the
CXADRL1,
GCUD1, or RNF43 protein, and expressing the polynucleotide sequence. In
addition, the
present invention provides vectors comprising a nucleotide sequence encoding
the
CXADRL1, GCUD1, or RNF43 protein, and host cells harboring a polynucleotide
encoding the CXADRL1, GCUD1, or RNF43 protein. Such vectors and host cells may
be used for producing the CXADRL1, GCUD1, or RNF43 protein.
An antibody that recognizes the CXADRL1, GCUD1, or RNF43 protein is also
provided by the present application. In part, an antisense polynucleotide
(e.g., antisense
DNA), ribozyme, and siRNA (small interfering RNA) of the CXADRLI, GCUD1, or
RNF43 gene is also provided.
The present invention further provides a method for diagnosis of cell
proliferative
diseases that includes determining an expression level of the gene in
biological sample of
specimen, comparing the expression level of CXADRLI, GCUD1, orRNF43 gene with
that
in normal sample, and defining a high expression level of the CXADRLI, GCUD1,
or
RNF43 gene in the sample as having a cell proliferative disease such as
cancer. The
disease diagnosed by the expression level of CXADRLI or GCUD1 is suitably a
gastric,
colorectal, and liver cancer; and that detected by the expression level of
RNF43 is


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colorectal, lung, gastric, and liver cancer.
Further, a method of screening for a compound for treating a cell
proliferative
disease is provided. The method includes contacting the CXADRLI, GCUD1, or
RNF43
polypeptide with test compounds, and selecting test compounds that bind to the
CXADRL1, GCUD1, or RNF43 polypeptide.
The present invention further provides a method of screening for a compound
for
treating a cell proliferative disease, wherein the method includes contacting
the CXADRLI,
GCUD1, or RNF43 polypeptide with a test compound, and selecting the test
compound
that suppresses the expression level or biological activity of the CXADRL1,
GCUD1, or
RNF43 polypeptide.
Alternatively, the present invention provides a method of screening for a
compound
for treating a cell proliferative disease, wherein the method includes
contacting CXADRLI
and AIP1 in the presence of a test compound, and selecting the test compound
that inhibits
the binding of CXADRL1 and AIP1.
Furthermore, the present invention provides a method of screening for a
compound
for treating a cell proliferative disease, wherein the method includes
contacting RNF43 and
NOTCH2 or STRIN in the presence of a test compound, and selecting the test
compound
that inhibits the binding of RNF43 and NOTCH2 or STRIN.
The present application also provides a pharmaceutical composition for
treating cell
proliferative disease, such as cancer. The pharmaceutical composition may be,
for
example, an anti-cancer agent. The pharmaceutical composition can be described
as at
least a portion of the antisense S-oligonucleotides or siRNA of the CXADRLI,
GCUD1, or
RNF43 polynucleotide sequence shown and described in SEQ ID NO: 1, 3, or 5,
respectively. A suitable antisense S-oligonucleotide has the nucleotide
sequence selected
from the group of SEQ ID NO: 23, 25, 27, 29, or 31. The antisense S-
oligonucleotide of
CXADRLI including those having the nucleotide sequence of SEQ ID NO: 23 or 25
may
be suitably used to treat gastric cancer; the antisense S-oligonucleotide of
GCUD1
including those having the nucleotide sequence of SEQ ID NO: 27 or 29 suitably
to treat
gastric, colorectal, or liver cancer; and the antisense S-oligonucleotide of
RNF43 including
those having the nucleotide sequence of SEQ ID NO: 31 suitably for colorectal,
lung,
gastric, or liver cancer. A suitable siRNA consists of a set of nucleotides
with the
nucleotide sequences selected from the group of SEQ ID NOs: 40 and 41, 42 and
43, or 62
and 63. The siRNA of CXADRLI consisting of a set of nucleotides with the
nucleotide
sequence of SEQ ID NOs: 40 and 41, or 42 and 43 may be suitably used to treat
gastric,
colorectal, or liver cancer; and the siRNA of RNF43 consisting of a set of
nucleotides with
the nucleotide sequence of SEQ ID NOs: 62 and 63 suitably for colorectal,
lung, gastric, or


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liver cancer. The pharmaceutical compositions may be also those comprising the
compounds selected by the present methods of screening for compounds for
treating cell
proliferative diseases.
The course of action of the pharmaceutical composition is desirably to inhibit
growth of the cancerous cells. The pharmaceutical composition may be applied
to
mammals including humans and domesticated mammals.
The present invention further provides methods for treating a cell
proliferative
disease using the pharmaceutical composition provided by the present
invention.
In addition, the present invention provides method for treating or preventing
cancer,
which method comprises the step of administering the CXADRL1, GCUD1, or RNF43
polypeptide. It is expected that anti tumor immunity be induced by the
administration of
the CXADRLI, GCUD1, or RNF43 polypeptide. Thus, the present invention also
provides method for inducing anti tumor immunity, which method comprises the
step of
administering the CXADRL1, GCUD1, or RNF43 polypeptide, as well as
pharmaceutical
composition for treating or preventing cancer comprising the CXADRLI, GCUD1,
or
RNF43 polypeptide.
It is to be understood that both the foregoing summary of the invention and
the
following detailed description are of a preferred embodiment, and not
restrictive of the
invention or other alternate embodiments of the invention.
Brief Description of the Drawings
Fig. la to 1d depict the expression of A5928 (CXADRL1) and C8121(GCUD1) in
gastric cancers. Fig. la depicts the relative expression ratios (cancer/non-
cancer) of
A5928 in primary 14 gastric cancers examined by cDNA microarray. Its
expression was
up-regulated (Cy3:Cy5 intensity ratio, >2.0) in 14 of the 14 gastric cancers
that passed
through the cutoff filter (both Cy3 and Cy5 signals greater than 25,000). Fig.
lb depicts
the relative expression ratios (cancer/non-cancer) of C8121 in primary 12
gastric cancers
examined by cDNA microarray. Its expression was up-regulated (Cy3:Cy5
intensity ratio,
>2.0) in 10 of the 12 gastric cancers that passed through the cutoff filter.
Fig 1c depicts
the expression of CXADRL1 analyzed by semi-quantitative RT-PCR using 10
gastric
cancer cases. Fig. 1d depicts the expression of GCUD1 analyzed by semi-
quantitative
RT-PCR using 9 gastric cancer cases. Expression of GAPDH served as an internal
control for both the expression analyses of CXADRL1 and GCUD1.
Fig. 2a and 2b depict the expression of CXADRL1 in various human tissues and
the
predicted protein structure and protein motifs of CXADRLI. Fig. 2a is a
photograph
depicting expression of CXADRL1 in various human tissues analyzed by multiple-
tissue


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northern blot analysis. Fig. 2b depicts the predicted protein structure of
CXADRL1.
The CXADRLI cDNA consists of 3,423 nucleotides with an ORF of 1,296
nucleotides and
is composed of 7 exons.
Fig. 3a to 3c depict the growth-promoting effect of CXADRL1. Fig. 3a is a
5 photograph depicting the result of colony formation assays of NIH3T3 cells
transfected
with CXADRLI. Fig. 3b depicts the expression of exogeneous CXADRL1 in
NIH3T3-CXADRL1 cells analyzed by semi-quantitative RT-PCR. Expression of
GAPDH served as an internal control. #2, #5, #6, and #7 all indicate NIH3T3
cells
transfected with CXADRL1. Fig. 3c depicts the number of NIH3T3 cells. Growth
of
10 NIH3T3-CXADRL1 cells was statistically higher than that of mock (NIH3T3-
LacZ) cells
in culture media containing 10% FBS (P<0.05).
Fig. 4 depicts the growth-inhibitory effect of antisense S-oligonucleotides
designated to suppress CXADRL1 in MKN-1 cells. CXADRL1-AS4 and
CXADRL1-AS5 were demonstrated to suppress the growth of MKN-1 cells.
Fig. 5A to 5C depict the growth suppressive effect of CXADRL1-siRNA on St-4
cells. Fig. 5A presents photographs depicting the expression of CXADRLI and
GAPDH
(control) in St-4 cells transfected with mock or CXADRL1-siRNA#7. Fig. 5B
depicts
photographs depicting the result of Giemsa's staining of viable cells treated
with
control-siRNA or CXADRL1-siRNA#7. Fig. 5C depicts the result of MTT assay on
cells
transfected with control plasmid or plasmids expressing CXADRL1-siRNA7.
Fig.6 depicts a photograph demonstrating the result of immunoblot analysis of
cells
expressing exogeneous Flag-tagged CXADRL1 protein with anti-CXADRL1 antisera
or
ant-Flag antibody.
Fig. 7 depicts the interaction between CXADRL1 and AIP1 examined by yeast
two-hybrid system. Fig. 7 is a photograph depicting the interaction of CXADRLI
with
AIP1 examined by the two-hybrid system.
Fig. 8 depicts the peptide specific cytotoxicity of CTL line raised by
CXADRL1-207 stimulation. The CTL line showed high cytotoxic activity on target
cells
(T2) pulsed with CXADRL1-207, whereas no significant cytotoxic activity was
detected
on the same target cells (T2) pulsed without peptides.
Fig. 9 depicts the cytotoxic activity of CXADRL1-207 CTL Clone on SNU475,
MKN74, and SNU-C4. CXADRL1-207 CTL Clone showed high cytotoxic activity on
SNU475 that expresses both CXADRL1 and HLA-A*0201. On the other hand,
CXADRL1-207 CTL Clone showed no significant cytotoxic activity on MKN74, which
expresses CXADRL1 but not HLA-A*0201. Furthermore, this CTL Clone did not show
significant cytotoxic activity on SNU-C4, which expresses HLA-A*0201 but not


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CXADRLI.
Fig. 10 depicts the result of the cold target inhibition assay. CXADRL1207 CTL
Clone specifically recognizes CXADRL1-207 in an HLA A*0201 restricted manner.
SNU475 labeled with Na251Cr 04 was prepared as a hot target, while CXADRL1207
peptide-pulsed T2 (Peptide +) was used as a cold target (Inhibitors). E/T
ratio was fixed
to 20. The cytotoxic activity on SNU475 was inhibited by the addition of T2
pulsed with
the identical peptide, while almost no inhibition by the addition of T2
without peptide
pulse.
Fig. 11 depicts the result of the blocking assay showing the effect of
antibodies
raised against HLA-Class I, HLA-Class 11, CD4, and CD8 on the cytotoxic
activity of
CXADRL1-207 CTL Clone. CXADRL1-207 CTL Clone showed cytotoxic activity in
HLA-Class I and CD8 restricted manner. To examine the characteristics of CTL
clone
raised with CXADRL1 peptide, antibodies against HLA-Class I, HLA-Class II,
CD4, and
CD8 were tested for their ability to inhibit the cytotoxic activity. The
horizontal axis
reveals % inhibition of the cytotoxicity. The cytotoxicity of CTL clone on
SNU475
targets was significantly reduced when anti class I and CD8 antibodies were
used. This
result indicates that the CTL clone recognizes the CXADRL1 derived peptide in
a
HLA-Class I and CD8 dependant manner.
Fig. 12 is a photograph depicting the result of Northern blot analysis of
GCUD1 in
various human tissues. The transcript of GCUD1 is approximately 3.5-kb by
size.
Fig. 13 shows a photograph depicting the subcellular localization of GCUD 1
observed by immunocytochemistry of cells transfected with pcDNA3.lmyc/His-
GCUD1.
cMyc-tagged GCUD 1 protein expressed from the plasmid localized in the
cytoplasm.
Fig. 14 is a photograph showing the growth-promoting effect of GCUD 1 on
NIH3T3 cells examined by colony formation assays.
Fig. 15 depicts the growth inhibitory effect of antisense S-oligonucleotides
designated to suppress GCUD1 on MKN28 cells. GCUDl AS5 and GCUDl AS8 were
revealed to suppress the growth of MKN-28 cells.
Fig. 16 depicts a photograph showing the purification of recombinant GCUD1
protein.
Fig. 17 depicts a photograph demonstrating the result of immunoblot analysis
of
cells expressing exogenous Flag-tagged GCUD1 protein with anti-GCUD1 antisera
or
ant Flag antibody.
Fig. 18A and 18B depict the peptide specific cytotoxicity of CTL line raised
by
GCUD 1-196 (A) or GCUD 1 272 (B) stimulation. The CTL line showed high
cytotoxic
activity on target cells (T2) pulsed with GCUD 1-196 or GCUD 1 272, whereas no

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significant cytotoxic activity

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was detected on the same target cells (T2) pulsed without peptides.
Fig. 19 depicts the cytotoxic activity of GCUD1-196 CTL Clone on SNU475 and
MKN45. GCUD1-196 CTL Clone showed high cytotoxic activity on SNU475 that
expresses both GCUD1 and HLA-A*0201. On the other hand, GCUD1-196 CTL Clone
showed no significant cytotoxic activity on MKN45, which expresses GCUD1 but
not
HLA-A*0201.
Fig. 20 depicts the result of the cold target inhibition assay. GCUD1-196 CTL
Clone specifically recognizes GCUD1-196 in an HLA-A*0201 restricted manner.
SNU475 labeled with Na251Cr 04 was prepared as a hot target, while GCUD1-196
peptide-pulsed T2 (Peptide +) was used as a cold target (Inhibitors). E/T
ratio was fixed
to 20. The cytotoxic activity on SNU475 was inhibited by the addition of T2
pulsed with
the identical peptide, while almost no inhibition by the addition of T2
without peptide
pulse.
Fig. 21 depicts the result of the blocking assay showing the effect of
antibodies
raised against HLA-Class I, HLA-Class II, CD4, and CD8 on the cytotoxic
activity of
GCUD1-196 CTL Clone. GCUD1-196 CTL Clone showed cytotoxic activity in
HLA-Class I and CD8 restricted manner. To examine the characteristics of CTL
clone
raised with GCUD1 peptide, antibodies against HLA-Class I, HLA-Class II, CD4,
and
CD8 were tested for their ability to inhibit the cytotoxic activity. The
horizontal axis
reveals % inhibition of the cytotxicity. The cytotoxicity of CTL clone on
SNU475 targets
was significantly reduced when anti class I and CD8 antibodies were used. This
result
indicates that the CTL clone recognizes the GCUD1 derived peptide in a HLA
Class I and
CD8 dependent manner.
Fig. 22a and 22b depict the expression of FL120315 in colon cancer. Fig. 22a
depicts the relative expression ratios (cancer/non-cancer) of FLJ20315 in 11
primary colon
cancer cases examined by cDNA microarray. Its expression was up-regulated
(Cy3:Cy5
intensity ratio, >2.0) in 10 of the 11 colon cancer cases that passed through
the cut-off filter
(both Cy3 and Cy5 signals greater than 25,000). Fig. 22b depicts the
expression of
FLJ20315 analyzed by semi-quantitative RT-PCR using additional 18 colon cancer
cases
(T, tumor tissue; N, normal tissue). Expression of GAPDH served as an internal
control.
Fig. 23a depicts a photograph showing the result of fetal-tissue northern blot
analysis of R1VF43 in various human fetal tissues. Fig. 23b depicts the
predicted protein
structure of RNF43.
Fig. 24a and 24b show photographs depicting the subcellular localization of
myc-tagged RNF43 protein. Fig. 24a is a photograph depicting the result of
Western-blot
analysis of myc-tagged RNF43 protein using extracts from COST cells
transfected with


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either pcDNA3.1-myc/His-RNF43 or control plasmids (mock). Fig. 24b presents
photographs of the transfected cells that were stained with mouse anti-myc
antibody and
visualized by FITC-conjugated secondary antibody. Nuclei were counter-stained
with
DAPI.
Fig. 25a to 25c depicts the effect of RNF43 on cell growth. Fig. 25a is a
photograph
depicting the result of colony formation assay of RNF43 in NIH3T3 cells. Fig.
25b
presents photographs depicting the expression of RNF43 in mock (COS7-pcDNA)
and
COS7-RNF43 cells that was established by the transfection of COST cells with
pcDNA-RNF43. Fig. 25c depicts the result of comparison on cell growth between
COS7-RNF43 cells stably expressing exogenous RNF43 and mock cells.
Fig. 26a and 26b depict the growth-inhibitory effect of antisense S-
oligonucleotides
designed to suppress RNF43. Fig. 26a presents photographs depicting the
expression of
RNF43 in LoVo cells treated for 12 h with either control (RNF43-S1) or
antisense
S-oligonucleotides (RNF43-AS1) analyzed by semi-quantitative RT-PCR. Fig. 26b
depicts the cell viability of LoVo cells after treatment with the control or
antisense
S-oligonucleotides measured by MTT assay. The MTT assay was carried out in
triplicate.
Fig. 27A to 27C depict the growth suppressive effect of RNF43-siRNAs. Fig.
27A presents photographs depicting the effect of RNF43-siRNAs on the
expression of
RNF43. Fig. 27B presents photographs depicting the result of Giemsa's staining
of
viable cells after the treatment with control- siRNA or RNF43-siRNAs. Fig. 27C
depicts
the result of MTT assay on cells transfected with control plasmid or plasmids
expressing
RNF43-siRNAs. *, a significant difference (p <0.05) as determined by a
Fisher's
protected least significant difference test.
Fig. 28A and 28B depict the expression of tagged RNF43 protein. Fig. 28A is a
photograph depicting the result of Western-blot analysis of Flag-tagged RNF43
protein
secreted in the culture media of COST cells transfected with pFLAG-5CMV-RNF43
(lane
2) or mock vector (lane 1). Fig. 28B is a photograph depicting the result of
Western-blot
analysis of Myc-tagged RNF43 protein secreted in the culture media of COST
cells
transfected with pcDNA3.1-Myc/His-RNF43 (lane 2) or mock vector (lane 1).
Fig. 29A and 29B depict the growth promoting effect of conditioned media
containing the Myc-tagged or Flag-tagged RNF43 protein. Fig. 29A presents
photographs depicting the morphology of NIH3T3 cells cultured in control media
(1) or in
conditioned media of COST cells transfected with mock vector (2),
pcDNA3.1-Myc/His-RNF43 (3), or pFLAG-5CMV-RNF43 (4). Fig. 29B depicts the
number of NIH3T3 cells cultured in the indicated media described in Fig. 29A.
Data are


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shown as means of triplicate experiments for each group; bars, SE. *,
significant
difference when compared with control, mock(p<0.05).
Fig. 30A to 30C depict the preparation of N-terminal (N1) and C-terminal (C1)
recombinant protein of RNF43. Fig. 30A depicts the schematic structure of the
5 recombinant protein RNF43-N1 and -C1. Fig. 30B is a photograph depicting the
expression of Nus-tagged RNF43-N1 protein in E. coli with (lane2) or without
(lane 1)
0.2 mM of IPTG. Fig. 30C is a photograph depicting the expression of NusTM-
tagged
RNF43-C1 protein in E. coli with (lane2) or without (lane 1) 1mM of IPTG.
Fig. 31A and 31B depict the interaction between RNF43 and NOTCH2 examined
10 by yeast two-hybrid system. Fig. 31A depicts the predicted structure and
the interacting
region of NOTCH2. (a) shows the predicted full length structure of NOTCH2
protein,
and (b) shows the predicted responsible region for the interaction (ECD,
Extracellular
domain; TM, transmembrane domain; ICD, Intracellular domain). Fig. 31B is a
photograph depicting the interaction of RNF43 with NOTCH2 examined by the two-
hybrid
15 system.
Fig. 32A and 32B depict the interaction between RNF43 and STRIN examined by
the yeast two-hybrid system. Fig. 32A depicts the predicted structure and the
interacting
region of STRIN. (a) shows the predicted full length structure of STRIN
protein, and (b)
shows the predicted responsible region for the interaction (RING, RING
domain). Fig.
32B is a photograph depicting the interaction of RNF43 with STRIN examined by
the
two-hybrid system.
Fig. 33 depicts the peptide specific cytotoxicity of CTL line raised by RNF43-
721
stimulation. The CTL line showed high cytotoxic activity on target cells
(TISI) pulsed
with RNF43-721 (quadrilateral line), whereas no significant cytotoxic activity
was
detected on the same target cells (TISI) pulsed without peptides (triangular
line). CTL
line was demonstrated to have a peptide specific cytotoxicity.
Fig. 34 depicts the peptide specific cytotoxicity of CTL clones raised by
RNF43-721 stimulation. The cytotoxic activity of 13 RNF43-721 CTL clones on
peptide-pulsed targets (TISI) was tested as described in the Materials and
Methods. The
established RNF43-721 CTL clones had very potent cytotoxic activity on target
cells
(TISI) pulsed with the peptides without showing any significant cytotoxic
activity on the
same target cells (TISI) that were not pulsed with any peptides.
Fig. 35 depicts the cytotoxic activity of RNF43-721 CTL Clone 45 on HT29,
WiDR and HCT116. RNF43-721 CTL Clone recognizes and lyses tumor cells that
endogenously express RNF43 in an HLA restricted fashion. HT29, WiDR and HCT116
all endogenously express RNF43, and RNF43-721 CTL Clone 45 served as an
effector cell.


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TISI was used as the target that does not express RNF43. RNF43-721 CTL Clone
45
showed high cytotoxic activity on HT29 (filled triangular line) and WiDR
(diamond line)
that express both RNF43 and HLA-A24. On the other hand, RNF43-721 CTL Clone 45
showed no significant cytotoxic activity on HCT1 16 (empty triangular line),
which
expresses RNF43 but not HLA-A24, and TISI (empty quadrilateral line), which
expresses
HLA-A24 but not RNF43. Moreover, RNF43-721 CTL Clone 45 showed no cytotoxic
activity on irrelevant peptide pulsed TISI (filled quadrilateral dotted line)
and SNU-C4
(filled circle line) which expresses RNF43 but little HLA-A24.
Fig. 36 depicts the result of the cold target inhibition assay. RNF43-721 CTL
Clone specifically recognizes RNF 43-721 in an HLA-A24 restricted manner. HT29
labeled with Na251Cr 04 was prepared as a hot target, while RNF43-721 peptide-
pulsed
TISI (Peptide +) was used as a cold target (Inhibitors). E/T ratio was fixed
to 20. The
cytotoxic activity on HT29 was inhibited by the addition of TISI pulsed with
the identical
peptide (filled quadrilateral line), while almost no inhibition occurred by
the addition of
TISI without peptide pulsing (empty quadrilateral line).
Fig. 37 depicts the result of the blocking assay showing the effect of
antibodies
raised against HLA-Class I, HLA-Class II, CD3, CD4, and CD8 on the cytotoxic
activity
of RNF43-721 CTL Clone. RNF43-721 CTL Clone showed cytotoxic activity in
HLA-Class I, CD3 and CD8 restricted manner. To examine the characteristics of
CTL
clone raised with RNF43 peptide, antibodies against HLA-Class I, HLA-Class II,
CD3,
CD4, and CD8 were tested for their ability to inhibit the cytotoxic activity.
The
horizontal axis reveals % inhibition of the cytotoxicity. The cytotoxicity of
CTL clone on
WiDR targets was significantly reduced when anti class I, CD3, and CD8
antibodies were
used. This result indicates that the CTL clone recognizes the RNF43 derived
peptide in a
HLA Class I, CD3 and CD8 dependent manner.
Fig. 38A and 38B depict the peptide specific cytotoxicity of the CTL lines
raised
with RNF43-11-9 (A) or RNF43-11-10 (B). These CTL lines showed high cytotoxic
activity on target cells (T2) pulsed with RNF43-11-9 or RNF43-11-10, whereas
no
significant cytotoxic activity was observed on the same target cells (T2)
pulsed without
peptides.
Fig. 39A and 39B depict the peptide specific cytotoxicity of CTL clones raised
by
RNF43-11-9 (A) or RNF43-11-10 (B) stimulation. Cytotoxic activity of 4 RNF43-
11-9
CTL clones or 7 RNF43-11-10 clones on peptide-pulsed targets (T2) was tested
as
described in the Materials and Methods. The established RNF43-11 9 and RNF43-
11-10
CTL clones had very potent cytotoxic activities on target cells (T2) pulsed
with the
peptides without showing any significant cytotoxic activity on the same target
cells (T2)

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that were not pulsed with any peptides.

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Fig. 40A and 40B depict the cytotoxic activity of RNF43-5 CTL Clone 90 and
RNF43-17 CTL Clone 25 on HT29 and DLD-1. RNF43-5 CTL Clone 90 and RNF43-17
CTL Clone 25 recognize and lyses tumor cells that endogenously express RNF43
in an
HLA restricted fashion. HT29 and DLD-1 all endogenously express RNF43, and
RNF43-5 CTL Clone 90 and RNF43-17 CTL Clone 25 served as an effector cell. T2
was
used as the target that does not express RNF43. RNF43-5 CTL Clone 90 and RNF43-
17
CTL Clone 25 showed high cytotoxic activity on DLD-1 that express both RNF43
and
HLA A*0201. On the other hand, RNF43-5 CTL Clone 90 and RNF43-17 CTL Clone 25
showed no significant cytotoxic activity on HT29, which expresses RNF43 but
not
HLA A*0201.
Fig. 41 depicts the result of cold target inhibition assay. RNF43-11-9 CTL
Clone
specifically recognizes RNF 43-11-9 in a HLA-A2 restricted manner. HCT116
labeled
with Na25'Cr 04 was prepared as a hot target, while RNF43-119 peptide-pulsed
T2
(Peptide +) was used as a cold target (Inhibitors). E/T ratio was fixed to 20.
The
cytotoxic activity on HCT116 was inhibited by the addition of T2 pulsed with
the identical
peptide, while almost no inhibition observed by the addition of TISI without
peptide pulse.
Detailed Description of the Invention
The words "a", "an", and "the" as used herein mean "at least one" unless
otherwise
specifically indicated.
The present application identifies novel human genes CXADRL1 and GCUD1 whose
expression is markedly elevated in gastric cancer compared to corresponding
non-cancerous tissues. The CXADRLI cDNA consists of 3423 nucleotides that
contain
an open reading frame of 1296 nucleotides as set forth in SEQ ID NO: 1. The
open
reading frame encodes a putative 431-amino acid protein. CXADRLI associates
with
AIP 1. AIP 1 (atripin-1 -interacting protein 1) is a protein that associates
with atropin-1, a
gene responsible fora hereditary disease, dentatorubral- pallidoluysian
atrophy. AIP1
encodes a deduced 1455-amino acid protein conatining guanylate kinase-like
domain, two
WW domains and five PDZ domains. The mouse homolog of AIP1 was shown to
interact with activin type IIA. However, the function of AIP1 remains to be
resolved.
The predicted amino acid sequence showed an identity of about 37% to CXADR
(coxsackie and adenovirus receptor). Therefore this protein was dubbed CXADRL1
(coxsackie and adenovirus receptor like 1). On the other hand, the GCUD1 cDNA
consists of 4987 nucleotides that contain an open reading frame of 1245
nucleotides as set
forth in SEQ ID NO: 3. The open reading frame encodes a putative 414-amino
acid

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protein. Since the expression of the protein was up-regulated in gastric
cancer, the
protein was dubbed GCUD1 (up-regulated in gastric cancer).
Furthermore, the present invention encompasses novel human gene RNF43 whose
expression is markedly elevated in colorectal cancer compared to corresponding
non-cancerous tissue. The RNF43 cDNA consists of 5345 nucleotides that contain
an
open reading frame of 2352 nucleotides as set forth in SEQ ID NO: 5. The open
reading
frame encodes a putative 783-amino acid protein. RNF43 associates with NOTCH2
and
STRIN. NOTCH2 is reported as a large transmembrane receptor protein that is a
component of an evolutionarily conserved intercellular signaling mechanism.
NOTCH2
is a protein member of the Notch signaling pathway and is reported to be
involved in
glomerulogenesis in the kidney and development of heart and eye vasculature.
Furthermore, three Delta/Serrate/Lag-2 (DSL) proteins, Deltal, Jaggaedl, and
Jaggaed2,
are reported as functional ligands for NOTCH2. STRIN encodes a putative
protein that
shares 79% identity with mouse Trif. The function of STRIN or Trif remains to
be
clarified.
Consistently, exogenous expression of CXADRLI, GCUD1, or RNF43 into cells
conferred increased cell growth, while suppression of its expression with
antisense
S-oligonucleotides or small interfering RNA (siRNA) resulted in significant
growth-inhibition of cancerous cells. These findings suggest that CXADRL1,
GCUD1,
and RNF43 render oncogenic activities to cancer cells, and that inhibition of
the activity of
these proteins could be a promising strategy for the treatment of cancer.
The present invention encompasses novel human gene CXADRLJ, including a
polynucleotide sequence as described in SEQ ID NO: 1, as well as degenerates
and
mutants thereof, to the extent that they encode a CXADRL1 protein, including
the amino
acid sequence set forth in SEQ ID NO: 2 or its functional equivalent. Examples
of
polypeptides functionally equivalent to CXADRL1 include, for example,
homologous
proteins of other organisms corresponding to the human CXADRLI protein, as
well as
mutants of human CXADRL 1 proteins.
The present invention also encompasses novel human gene GCUD1, including a
polynucleotide sequence as described in SEQ ID NO: 3, as well as degenerates
and
mutants thereof, to the extent that they encode a GCUD1 protein, including the
amino acid
sequence set forth in SEQ ID NO: 4 or its functional equivalent. Examples of
polypeptides functionally equivalent to GCUD1 include, for example, homologous
proteins of other organisms corresponding to the human GCUD 1 protein, as well
as
mutants of human GCUD1 proteins.
Furthermore, the present invention encompasses novel human gene RNF43,


CA 02488404 2004-12-03
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including a polynucleotide sequence as described in SEQ ID NO: 5, as well as
degenerates
and mutants thereof, to the extent that they encode a RNF43 protein, including
the amino
acid sequence set forth in SEQ ID NO: 6 or its functional equivalent. Examples
of
polypeptides functionally equivalent to RNF43 include, for example, homologous
proteins
5 of other organisms corresponding to the human RNF43 protein, as well as
mutants of
human RNF43 proteins.
In the present invention, the term "functionally equivalent" means that the
subject
polypeptide has the activity to promote cell proliferation like CXADRL1,
GCUD1, or
RNF43 protein and to confer oncogenic activity to cancer cells. Whether the
subject
10 polypeptide has a cell proliferation activity or not can be judged by
introducing the DNA
encoding the subject polypeptide into a cell expressing the respective
polypeptide, and
detecting promotion of proliferation of the cells or increase in colony
forming activity.
Such cells include, for example, NIH3T3 cells for CXADRL1 and GCUD1; and
NIH3T3
cells, SW480 cells, and COST cells for RNF43. Alternatively, whether the
subject
15 polypeptide is functionally equivalent to CXADRLI may be judged by
detecting its
binding ability to AIP1. Furthermore, whether the subject polypeptide is
functionally
equivalent to RNF43 may be judged by detecting its binding ability to NOTCH2
or
STRIN.
Methods for preparing polypeptides functionally equivalent to a given protein
are
20 well known by a person skilled in the art and include known methods of
introducing
mutations into the protein. For example, one skilled in the art can prepare
polypeptides
functionally equivalent to the human CXADRL1, GCUD1, or RNF43 protein by
introducing an appropriate mutation in the amino acid sequence of either of
these proteins
by site-directed mutagenesis (Hashimoto-Gotoh et al., Gene 152:271-5 (1995);
Zoller and
Smith, Methods Enzymol 100: 468-500 (1983); Kramer et al., Nucleic Acids Res.
12:9441-9456 (1984); Kramer and Fritz, Methods Enzymol 154: 350-67 (1987);
Kunkel,
Proc Natl Acad Sci USA 82: 488-92 (1985); Kunkel, Methods Enzymol 85: 2763-6
(1988)).
Amino acid mutations can occur in nature, too. The polypeptide of the present
invention
includes those proteins having the amino acid sequences of the human CXADRL1,
GCUD1, or RNF43 protein in which one or more amino acids are mutated, provided
the
resulting mutated polypeptides are functionally equivalent to the human
CXADRL1,
GCUD1, or RNF43 protein. The number of amino acids to be mutated in such a
mutant
is generally 10 amino acids or less, preferably 6 amino acids or less, and
more preferably 3
amino acids or less.
Mutated or modified proteins, proteins having amino acid sequences modified by
substituting, deleting, inserting, and/or adding one or more amino acid
residues of a certain


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21
amino acid sequence, have been known to retain the original biological
activity (Mark et
al., Proc Nall Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids
Res
10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-
13
(1982)).
The amino acid residue to be mutated is preferably mutated into a different
amino
acid in which the properties of the amino acid side-chain are conserved (a
process known
as conservative amino acid substitution). Examples of properties of amino acid
side
chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic
amino acids (R,
D, N, C, E, Q, E H, K, S, T), and side chains having the following functional
groups or
characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a
hydroxyl group
containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a
carboxylic
acid and amide containing side-chain (D, N, E, Q); a base containing side-
chain (R, K, H);
and an aromatic containing side-chain (H, F, Y, W). Note, the parenthetic
letters indicate
the one-letter codes of amino acids.
An example of a polypeptide to which one or more amino acids residues are
added
to the amino acid sequence of human CXADRL1, GCUD1, or RNF43 protein is a
fusion
protein containing the human CXADRL1, GCUD1, or RNF43 protein. Fusion proteins
are, fusions of the human CXADRL1, GCUD1, or RNF43 protein and other peptides
or
proteins, and are included in the present invention. Fusion proteins can be
made by
techniques well known to a person skilled in the art, such as by linking the
DNA encoding
the human CXADRL1, GCUD1, or RNF43 protein of the invention with DNA encoding
other peptides or proteins, so that the frames match, inserting the fusion DNA
into an
expression vector and expressing it in a host. There is no restriction as to
the peptides or
proteins fused to the protein of the present invention.
Known peptides that can be used as peptides that are fused to the protein of
the
present invention include, for example, FLAG (Hopp et al., Biotechnology 6:
1204-10
(1988)), 6xHis containing six His (histidine) residues, 1OxHis, Influenza
agglutinin (HA),
human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV tag, E-
tag,
SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag, Protein C
fragment, and the
like. Examples of proteins that may be fused to a protein of the invention
include GST
(glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin
constant region,
j3-galactosidase, MBP (maltose-binding protein), and such.
Fusion proteins can be prepared by fusing commercially available DNA, encoding
the fusion peptides or proteins discussed above, with the DNA encoding the
polypeptide of
the present invention and expressing the fused DNA prepared.
An alternative method known in the art to isolate functionally equivalent


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22
polypeptides is, for example, the method using a hybridization technique
(Sambrook et al.,
Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press (1989)).
One
skilled in the art can readily isolate a DNA having high homology with a whole
or part of
the DNA sequence encoding the human CXADRL1, GCUD1, or RNF43 protein (i.e.,
SEQ
ID NO: 1, 3, or 5), and isolate functionally equivalent polypeptides to the
human
CXADRL1, GCUD1, or RNF43 protein from the isolated DNA. The polypeptides of
the
present invention include those that are encoded by DNA that hybridize with a
whole or
part of the DNA sequence encoding the human CXADRL1, GCUD1, or RNF43 protein
and are functionally equivalent to the human CXADRL1, GCUD1, or RNF43 protein.
These polypeptides include mammal homologues corresponding to the protein
derived
from human (for example, a polypeptide encoded by a monkey, rat, rabbit and
bovine
gene). In isolating a cDNA highly homologous to the DNA encoding the human
CXADRL1 protein from animals, it is particularly preferable to use tissues
from testis or
ovary. Alternatively, in isolating a cDNA highly homologous to the DNA
encoding the
human GCUD1 from animals, it is particularly preferable to use tissues from
testis, ovary,
or brain. Further, in isolating a cDNA highly homologous to the DNA encoding
the
human RNF43 protein from animals, it is particularly preferable to use tissue
from fetal
lung or fetal kidney.
The condition of hybridization for isolating a DNA encoding a polypeptide
functionally equivalent to the human CXADRL1, GCUD1, or RNF43 protein can be
routinely selected by a person skilled in the art. For example, hybridization
may be
performed by conducting prehybridization at 68 C for 30 min or longer using
"Rapid-hyb
buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 C
for 1
hour or longer. The following washing step can be conducted, for example, in a
low
stringent condition. A low stringent condition is, for example, 42 C, 2X SSC,
0.1% SDS,
or preferably 50 C, 2X SSC, 0.1% SDS. More preferably, high stringent
conditions are
used. A high stringent condition is, for example, washing 3 times in 2X SSC,
0.01% SDS
at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37
C for 20
min, and washing twice in 1x SSC, 0.1% SDS at 50 C for 20 min. However,
several
factors, such as temperature and salt concentration, can influence the
stringency of
hybridization and one skilled in the art can suitably select the factors to
achieve the
requisite stringency.
In place of hybridization, a gene amplification method, for example, the
polymerase chain reaction (PCR) method, can be utilized to isolate a DNA
encoding a
polypeptide functionally equivalent to the human CXADRL1, GCUD1, or RNF43
protein,
using a primer synthesized based on the sequence information of the protein
encoding


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23
DNA (SEQ ID NO: 1, 3, or 5).
Polypeptides that are functionally equivalent to the human CXADRL1, GCUD1, or
RNF43 protein encoded by the DNA isolated through the above hybridization
techniques
or gene amplification techniques, normally have a high homology to the amino
acid
sequence of the human CXADRL1, GCUD1, or RNF43 protein. "High homology"
typically refers to a homology of 40% or higher, preferably 60% or higher,
more preferably
80% or higher, even more preferably 95% or higher. The homology of a
polypeptide can
be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad
Sci USA
80: 726-30 (1983)".
A polypeptide of the present invention may have variations in amino acid
sequence,
molecular weight, isoelectric point, the presence or absence of sugar chains,
or form,
depending on the cell or host used to produce it or the purification method
utilized.
Nevertheless, so long as it has a function equivalent to that of the human
CXADRL1,
GCUD1, or RNF43 protein of the present invention, it is within the scope of
the present
invention.
The polypeptides of the present invention can be prepared as recombinant
proteins
or natural proteins, by methods well known to those skilled in the art. A
recombinant
protein can be prepared by inserting a DNA, which encodes the polypeptide of
the present
invention (for example, the DNA comprising the nucleotide sequence of SEQ ID
NO: 1, 3,
or 5), into an appropriate expression vector, introducing the vector into an
appropriate host
cell, obtaining the extract, and purifying the polypeptide by subjecting the
extract to
chromatography, for example, ion exchange chromatography, reverse phase
chromatography, gel filtration, or affinity chromatography utilizing a column
to which
antibodies against the protein of the present invention is fixed, or by
combining more than
one of aforementioned columns.
Also when the polypeptide of the present invention is expressed within host
cells
(for example, animal cells and E. coli) as a fusion protein with glutathione-S-
transferase
protein or as a recombinant protein supplemented with multiple histidines, the
expressed
recombinant protein can be purified using a glutathione column or nickel
column.
Alternatively, when the polypeptide of the present invention is expressed as a
protein
tagged with c-myc, multiple histidines, or FLAG it can be detected and
purified using
antibodies to c-myc, His, or FLAG respectively.
After purifying the fusion protein, it is also possible to exclude regions
other than
the objective polypeptide by cutting with thrombin or factor-Xa as required.
A natural protein can be isolated by methods known to a person skilled in the
art,
for example, by contacting the affinity column, in which antibodies binding to
the


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24
CXADRL1, GCUD1, or RNF43 protein described below are bound, with the extract
of
tissues or cells expressing the polypeptide of the present invention. The
antibodies can be
polyclonal antibodies or monoclonal antibodies.
The present invention also encompasses partial peptides of the polypeptide of
the
present invention. The partial peptide has an amino acid sequence specific to
the
polypeptide of the present invention and consists of at least 7 amino acids,
preferably 8
amino acids or more, and more preferably 9 amino acids or more. The partial
peptide can
be used, for example, for preparing antibodies against the polypeptide of the
present
invention, screening for a compound that binds to the polypeptide of the
present invention,
and screening for accelerators or inhibitors of the polypeptide of the present
invention.
A partial peptide of the invention can be produced by genetic engineering, by
known methods of peptide synthesis, or by digesting the polypeptide of the
invention with
an appropriate peptidase. For peptide synthesis, for example, solid phase
synthesis or
liquid phase synthesis may be used.
Furthermore, the present invention provides polynucleotides encoding the
polypeptide of the present invention. The polynucleotides of the present
invention can be
used for the in vivo or in vitro production of the polypeptide of the present
invention as
described above, or can be applied to gene therapy for diseases attributed to
genetic
abnormality in the gene encoding the protein of the present invention. Any
form of the
polynucleotide of the present invention can be used so long as it encodes the
polypeptide
of the present invention, including mRNA, RNA, cDNA, genomic DNA, chemically
synthesized polynucleotides. The polynucleotide of the present invention
include a DNA
comprising a given nucleotide sequences as well as its degenerate sequences,
so long as the
resulting DNA encodes a polypeptide of the present invention.
The polynucleotide of the present invention can be prepared by methods known
to
a person skilled in the art. For example, the polynucleotide of the present
invention can
be prepared by: preparing a cDNA library from cells which express the
polypeptide of the
present invention, and conducting hybridization using a partial sequence of
the DNA of the
present invention (for example, SEQ ID NO: 1, 3, or 5) as a probe. A cDNA
library can
be prepared, for example, by the method described in Sambrook et al.,
Molecular Cloning,
Cold Spring Harbor Laboratory Press (1989); alternatively, commercially
available cDNA
libraries maybe used. A cDNA library can be also prepared by: extracting RNAs
from
cells expressing the polypeptide of the present invention, synthesizing oligo
DNAs based
on the sequence of the DNA of the present invention (for example, SEQ ID NO:
1, 3, or 5),
conducting PCR using the oligo DNAs as primers, and amplifying cDNAs encoding
the
protein of the present invention.


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In addition, by sequencing the nucleotides of the obtained cDNA, the
translation
region encoded by the cDNA can be routinely determined, and the amino acid
sequence of
the polypeptide of the present invention can be easily obtained. Moreover, by
screening
the genomic DNA library using the obtained cDNA or parts thereof as a probe,
the
5 genomic DNA can be isolated.
More specifically, mRNAs may first be prepared from a cell, tissue, or organ
(e.g.,
testis or ovary for CXADRL1; testis, ovary, or brain for GCUD1; and fetal
lung, or fetal
kidney for RNF43) in which the object polypeptide of the invention is
expressed. Known
methods can be used to isolate mRNAs; for instance, total RNA may be prepared
by
10 guanidine ultracentrifugation (Chirgwin et al., Biochemistry 18:5294-9
(1979)) or AGPC
method (Chomczynski and Sacchi, Anal Biochem 162:156-9 (1987)). In addition,
mRNA
may be purified from total RNA using mRNA Purification Kit (Pharmacia) and
such or,
alternatively, mRNA may be directly purified by QuickPrep mRNA Purification
Kit
(Pharmacia).
15 The obtained mRNA is used to synthesize cDNA using reverse transcriptase.
cDNA may be synthesized using a commercially available kit, such as the AMV
Reverse
Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).
Alternatively, cDNA
may be synthesized and amplified following the 5'-RACE method (Frohman et al.,
Proc
Natl Acad Sci USA 85: 8998-9002 (1988); Belyavsky et al., Nucleic Acids Res
17:
20 2919-32 (1989)), which uses a primer and such, described herein, the 5'-
Ampli FINDER
RACE Kit (Clontech), and polymerase chain reaction (PCR).
A desired DNA fragment is prepared from the PCR products and ligated with a
vector DNA. The recombinant vectors are used to transform E. coli and such,
and a
desired recombinant vector is prepared from a selected colony. The nucleotide
sequence
25 of the desired DNA can be verified by conventional methods, such as
dideoxynucleotide
chain termination.
The nucleotide sequence of a polynucleotide of the invention may be designed
to
be expressed more efficiently by taking into account the frequency of codon
usage in the
host to be used for expression (Grantham et al., Nucleic Acids Res 9: 43-74
(1981)). The
sequence of the polynucleotide of the present invention may be altered by a
commercially
available kit or a conventional method. For instance, the sequence may be
altered by
digestion with restriction enzymes, insertion of a synthetic oligonucleotide
or an
appropriate polynucleotide fragment, addition of a linker, or insertion of the
initiation
codon (ATG) and/or the stop codon (TAA, TGA, or TAG).
Specifically, the polynucleotide of the present invention encompasses the DNA
comprising the nucleotide sequence of SEQ ID NO: 1, 3, or 5.


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26
Furthermore, the present invention provides a polynucleotide that hybridizes
under
stringent conditions with a polynucleotide having a nucleotide sequence of SEQ
ID NO: 1,
3, or 5, and encodes a polypeptide functionally equivalent to the CXADRL1,
GCUD1, or
RNF43 protein of the invention described above. One skilled in the art may
appropriately
choose stringent conditions. For example, low stringent condition can be used.
More
preferably, high stringent condition can be used. These conditions are the
same as that
described above. The hybridizing DNA above is preferably a cDNA or a
chromosomal
DNA.
The present invention also provides a vector into which a polynucleotide of
the
present invention is inserted. A vector of the present invention is useful to
keep a
polynucleotide, especially a DNA, of the present invention in host cell, to
express the
polypeptide of the present invention, or to administer the polynucleotide of
the present
invention for gene therapy.
When E. coli is a host cell and the vector is amplified and produced in a
large
amount in E. coli (e.g., JM109, DH5a, HB101, or XL1Blue), the vector should
have "ori"
to be amplified in E. coli and a marker gene for selecting transformed E. coli
(e.g., a
drug-resistance gene selected by a drug such as ampicillin, tetracycline,
kanamycin,
chloramphenicol or the like). For example, M13-series vectors, pUC-series
vectors,
pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T,
pDIRECT, and
pT7 can also be used for subcloning and extracting cDNA as well as the vectors
described
above. When a vector is used to produce the protein of the present invention,
an
expression vector is especially useful. For example, an expression vector to
be expressed
in E. coli should have the above characteristics to be amplified in E. coli.
When E. coli,
such as JM109, DH5a, HB101, or XL1 Blue, are used as a host cell, the vector
should have
a promoter, for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989);
FASEB J
6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)),
or T7
promoter or the like, that can efficiently express the desired gene in E.
coli. In that
respect, pGEX-5X-1 (Pharmacia), "QlAexpress system" (Qiagen), pEGFP and pET
(in this
case, the host is preferably BL21 which expresses T7 RNA polymerase), for
example, can
be used instead of the above vectors. Additionally, the vector may also
contain a signal
sequence for polypeptide secretion. An exemplary signal sequence that directs
the
polypeptide to be secreted to the periplasm of the E. coli is the pelB signal
sequence (Lei et
al., J Bacteriol 169: 4379 (1987)). Means for introducing of the vectors into
the target
host cells include, for example, the calcium chloride method, and the
electroporation
method.
In addition to E. coli, for example, expression vectors derived from mammals
(for


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27
example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18(17): 5322
(1990)),
pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac-
to-BAC
baculovirus expression system" (GIBCO BRL), pBacPAK8), expression vectors
derived
from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses
(e.g.,
pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g.,
pZlpneo),
expression vector derived from yeast (e.g., "Pichia Expression Kit"
(Invitrogen), pNV11,
SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608,
pKTH50)
can be used for producing the polypeptide of the present invention.
In order to express the vector in animal cells, such as CHO, COS, or NIH3T3
cells,
the vector should have a promoter necessary for expression in such cells, for
example, the
SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR
promoter, the
EF1a promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV
promoter,
and the like, and preferably a marker gene for selecting transformants (for
example, a drug
resistance gene selected by a drug (e.g., neomycin, G418)). Examples of known
vectors
with these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV,
pOPRSV, and pOP13.
In addition, methods may be used to express a gene stably and, at the same
time, to
amplify the copy number of the gene in cells. For example, a vector comprising
the
complementary DHFR gene (e.g., pCHO I) may be introduced into CHO cells in
which the
nucleic acid synthesizing pathway is deleted, and then amplified by
methotrexate (MTX).
Furthermore, in case of transient expression of a gene, the method wherein a
vector
comprising a replication origin of SV40 (pcD, etc.) is transformed into COS
cells
comprising the SV40 T antigen expressing gene on the chromosome can be used.
Apolypeptide of the present invention obtained as above may be isolated from
inside or outside (such as medium) of host cells, and purified as a
substantially pure
homogeneous polypeptide. The term "substantially pure" as used herein in
reference to a
given polypeptide means that the polypeptide is substantially free from other
biological
macromolecules. The substantially pure polypeptide is at least 75% (e.g., at
least 80, 85,
95, or 99%) pure by dry weight. Purity can be measured by any appropriate
standard
method, for example by column chromatography, polyacrylamide gel
electrophoresis, or
HPLC analysis. The method for polypeptide isolation and purification is not
limited to
any specific method; in fact, any standard method may be used.
For instance, column chromatography, filter, ultrafiltration, salt
precipitation,
solvent precipitation, solvent extraction, distillation, immunoprecipitation,
SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis,
dialysis, and
recrystallization may be appropriately selected and combined to isolate and
purify the


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28
polypeptide.
Examples of chromatography include, for example, affinity chromatography,
ion-exchange chromatography, hydrophobic chromatography, gel filtration,
reverse phase
chromatography, adsorption chromatography, and such (Strategies for Protein
Purification
and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et
al., Cold
Spring Harbor Laboratory Press (1996)). These chromatographies may be
performed by
liquid chromatography, such as HPLC and FPLC. Thus, the present invention
provides
for highly purified polypeptides prepared by the above methods.
A polypeptide of the present invention may be optionally modified or partially
deleted by treating it with an appropriate protein modification enzyme before
or after
purification. Useful protein modification enzymes include, but are not limited
to, trypsin,
chymotrypsin, lysylendopeptidase, protein kinase, glucosidase, and so on.
Th.e present invention provides an antibody that binds to the polypeptide of
the
invention. The antibody of the invention can be used in any form, such as
monoclonal or
polyclonal antibodies, and includes antiserum obtained by immunizing an animal
such as a
rabbit with the polypeptide of the invention, all classes of polyclonal and
monoclonal
antibodies, human antibodies, and humanized antibodies produced by genetic
recombination.
A polypeptide of the invention used as an antigen to obtain an antibody may be
derived from any animal species, but preferably is derived from a mammal such
as a
human, mouse, or rat, more preferably from a human. A human-derived
polypeptide may
be obtained from the nucleotide or amino acid sequences disclosed herein.
According to the present invention, the polypeptide to be used as an
immunization antigen
may be a complete protein or a partial peptide of the protein. A partial
peptide may
comprise, for example, the amino (N)-terminal or carboxy (C)-terminal fragment
of a
polypeptide of the present invention. More specifically, a polypeptide of
CXADRL1
encompassing the codons from 235 to 276, from 493 to 537, or from 70 to 111
can be used
as partial peptides for producing antibodies against CXADRL1 of the present
invention.
Alternatively, for the production of antibodies against the polypeptide of the
present
invention, peptides comprising any one of following amino acid sequences may
be used.
- RNF43 ; SEQ ID No: 80, 97, or 108
- CXADRL1 ; SEQ ID No: 124
- GCUD1 ; SEQ ID No: 164
Herein, an antibody is defined as a protein that reacts with either the full
length or a
fragment of a polypeptide of the present invention.
A gene encoding a polypeptide of the invention or its fragment may be inserted
into


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29
a known expression vector, which is then used to transform a host cell as
described herein.
The desired polypeptide or its fragment may be recovered from the outside or
inside of
host cells by any standard method, and may subsequently be used as an antigen.
Alternatively, whole cells expressing the polypeptide or their lysates, or a
chemically
synthesized polypeptide may be used as the antigen.
Any mammalian animal may be immunized with the antigen, but preferably the
compatibility with parental cells used for cell fusion is taken into account.
In general,
animals of Rodentia, Lagomorpha, or Primates are used. Animals of Rodentia
include,
for example, mouse, rat, and hamster. Animals of Lagomorpha include, for
example,
rabbit. Animals of Primates include, for example, a monkey of Catarrhini (old
world
monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon, and
chimpanzees.
Methods for immunizing animals with antigens are known in the art.
Intraperitoneal injection or subcutaneous injection of antigens is a standard
method for
immunization of mammals. More specifically, antigens may be diluted and
suspended in
an appropriate amount of phosphate buffered saline (PBS), physiological
saline, etc. If
desired, the antigen suspension may be mixed with an appropriate amount of a
standard
adjuvant, such as Freund's complete adjuvant, made into emulsion, and then
administered
to mammalian animals. Preferably, it is followed by several administrations of
antigen
mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to
21 days.
An appropriate carrier may also be used for immunization. After immunization
as above,
serum is examined by a standard method for an increase in the amount of
desired
antibodies.
Polyclonal antibodies against the polypeptides of the present invention may be
prepared by collecting blood from the immunized mammal examined for the
increase of
desired antibodies in the serum, and by separating serum from the blood by any
conventional method. Polyclonal antibodies include serum containing the
polyclonal
antibodies, as well as the fraction containing the polyclonal antibodies may
be isolated
from the serum. Immunoglobulin G or M can be prepared from a fraction which
recognizes only the polypeptide of the present invention using, for example,
an affinity
column coupled with the polypeptide of the present invention, and further
purifying this
fraction using protein A or protein G column.
To prepare monoclonal antibodies, immune cells are collected from the mammal
immunized with the antigen and checked for the increased level of desired
antibodies in the
serum as described above, and are subjected to cell fusion. The immune cells
used for
cell fusion are preferably obtained from spleen. Other preferred parental
cells to be fused
with the above immunocyte include, for example, myeloma cells of mammalians,
and


CA 02488404 2004-12-03
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more preferably myeloma cells having an acquired property for the selection of
fused cells
by drugs.
The above immunocyte and myeloma cells can be fused according to known
methods, for example, the method of Milstein et al. (Galfre and Milstein,
Methods
5 Enzymol 73: 3-46 (1981)).
Resulting hybridomas obtained by the cell fusion may be selected by
cultivating
them in a standard selection medium, such as HAT medium (hypoxanthine,
aminopterin,
and thymidine containing medium). The cell culture is typically continued in
the HAT
medium for several days to several weeks, the time being sufficient to allow
all the other
10 cells, with the exception of the desired hybridoma (non-fused cells), to
die. Then, the
standard limiting dilution is performed to screen and clone a hybridoma cell
producing the
desired antibody.
In addition to the above method, in which a non-human animal is immunized with
an antigen for preparing hybridoma, human lymphocytes such as those infected
by EB
15 virus may be immunized with a polypeptide, polypeptide expressing cells, or
their lysates
in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma
cells
that are capable of indefinitely dividing, such as U266, to yield a hybridoma
producing a
desired human antibody that is able to bind to the polypeptide can be obtained
(Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
20 The obtained hybridomas are subsequently transplanted into the abdominal
cavity
of a mouse and the ascites are extracted. The obtained monoclonal antibodies
can be
purified by, for example, ammonium sulfate precipitation, a protein A or
protein G column,
DEAE ion exchange chromatography, or an affinity column to which the
polypeptide of
the present invention is coupled. The antibody of the present invention can be
used not
25 only for purification and detection of the polypeptide of the present
invention, but also as a
candidate for agonists and antagonists of the polypeptide of the present
invention. In
addition, this antibody can be applied to the antibody treatment for diseases
related to the
polypeptide of the present invention. When the obtained antibody is to be
administered to
the human body (antibody treatment), a human antibody or a humanized antibody
is
30 preferable for reducing immunogenicity.
For example, transgenic animals having a repertory of human antibody genes may
be immunized with an antigen selected from a polypeptide, polypeptide
expressing cells, or
their lysates. Antibody producing cells are then collected from the animals
and fused
with myeloma cells to obtain hybridoma, from which human antibodies against
the
polypeptide can be prepared (see W092-03918, W093-2227, W094-02602, W094-
25585,
W096-33735, and W096-34096).


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31
Alternatively, an immune cell, such as an immunized lymphocyte, producing
antibodies may be immortalized by an oncogene and used for preparing
monoclonal
antibodies.
Monoclonal antibodies thus obtained can be also recombinantly prepared using
genetic engineering techniques (see, for example, Borrebaeck and Larrick,
Therapeutic
Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers
LTD
(1990)). For example, a DNA encoding an antibody may be cloned from an immune
cell,
such as a hybridoma or an immunized lymphocyte producing the antibody,
inserted into an
appropriate vector, and introduced into host cells to prepare a recombinant
antibody. The
present invention also provides recombinant antibodies prepared as described
above.
Furthermore, an antibody of the present invention may be a fragment of an
antibody or modified antibody, so long as it binds to one or more of the
polypeptides of the
invention. For instance, the antibody fragment may be Fab, F(ab')2, Fv, or
single chain
Fv (scFv), in which Fv fragments from H and L chains are ligated by an
appropriate linker
(Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically,
an
antibody fragment may be generated by treating an antibody with an enzyme,
such as
papain or pepsin. Alternatively, a gene encoding the antibody fragment may be
constructed, inserted into an expression vector, and expressed in an
appropriate host cell
(see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and
Horwitz, Methods
Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515
(1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods
Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7
(1991)).
An antibody may be modified by conjugation with a variety of molecules, such
as
polyethylene glycol (PEG). The present invention provides for such modified
antibodies.
The modified antibody can be obtained by chemically modifying an antibody.
These
modification methods are conventional in the field.
Alternatively, an antibody of the present invention may be obtained as a
chimeric
antibody, between a variable region derived from nonhuman antibody and the
constant
region derived from human antibody, or as a humanized antibody, comprising the
complementarity determining region (CDR) derived from nonhuman antibody, the
frame
work region (FR) derived from human antibody, and the constant region. Such
antibodies
can be prepared by using known technology.
Antibodies obtained as above may be purified to homogeneity. For example, the
separation and purification of the antibody can be performed according to
separation and
purification methods used for general proteins. For example, the antibody may
be
separated and isolated by the appropriately selected and combined use of
column


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32
chromatographies, such as affinity chromatography, filter, ultrafiltration,
salting-out,
dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and
others
(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor
Laboratory (1988)), but are not limited thereto. A protein A column and
protein G
column can be used as the affinity column. Exemplary protein A columns to be
used
include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).
Exemplary chromatography, with the exception of affinity includes, for
example,
ion-exchange chromatography, hydrophobic chromatography, gel filtration,
reverse-phase
chromatography, adsorption chromatography, and the like (Strategies for
Protein
Purification and Characterization: A Laboratory Course Manual. Ed Daniel R.
Marshak et
al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic
procedures can
be carried out by liquid-phase chromatography, such as HPLC, and FPLC.
For example, measurement of absorbance, enzyme-linked immunosorbent assay
(ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or
immunofluorescence may be used to measure the antigen binding activity of the
antibody
of the invention. In ELISA, the antibody of the present invention is
immobilized on a
plate, a polypeptide of the invention is applied to the plate, and then a
sample containing a
desired antibody, such as culture supernatant of antibody producing cells or
purified
antibodies, is applied. Then, a secondary antibody that recognizes the primary
antibody
and is labeled with an enzyme, such as alkaline phosphatase, is applied, and
the plate is
incubated. Next, after washing, an enzyme substrate, such as p-nitrophenyl
phosphate, is
added to the plate, and the absorbance is measured to evaluate the antigen
binding activity
of the sample. A fragment of the polypeptide, such as a C-terminal or N-
terminal
fragment, may be used as the antigen to evaluate the binding activity of the
antibody.
BlAcore (Pharmacia) may be used to evaluate the activity of the antibody
according to the
present invention.
The above methods allow for the detection or measurement of the polypeptide of
the invention, by exposing the antibody of the invention to a sample assumed
to contain the
polypeptide of the invention, and detecting or measuring the immune complex
formed by
the antibody and the polypeptide.
Because the method of detection or measurement of the polypeptide according to
the invention can specifically detect or measure a polypeptide, the method may
be useful in
a variety of experiments in which the polypeptide is used.
The present invention also provides a polynucleotide which hybridizes with the
polynucleotide encoding human CXADRL1, GCUD1, or RNF43 protein (SEQ ID NO: 1,
3, or 5) or the complementary strand thereof, and which comprises at least 15
nucleotides.


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33
The polynucleotide of the present invention is preferably a polynucleotide
which
specifically hybridizes with the DNA encoding the polypeptide of the present
invention.
The term "specifically hybridize" as used herein, means that cross-
hybridization does not
occur significantly with DNA encoding other proteins, under the usual
hybridizing
conditions, preferably under stringent hybridizing conditions. Such
polynucleotides
include, probes, primers, nucleotides and nucleotide derivatives (for example,
antisense
oligonucleotides and ribozymes), which specifically hybridize with DNA
encoding the
polypeptide of the invention or its complementary strand. Moreover, such
polynucleotide
can be utilized for the preparation of DNA chip.
The present invention includes an antisense oligonucleotide that hybridizes
with
any site within the nucleotide sequence of SEQ ID NO: 1, 3, or 5. This
antisense
oligonucleotide is preferably against at least 15 continuous nucleotides of
the nucleotide
sequence of SEQ ID NO: 1, 3, or 5. The above-mentioned antisense
oligonucleotide,
which contains an initiation codon in the above-mentioned at least 15
continuous
nucleotides, is even more preferred. More specifically, such antisense
oligonucleotides
include those comprising the nucleotide sequence of SEQ ID NO: 23 or 25 for
suppressing
the expression of CXADRL1; SEQ ID NO: 27, or 29 for GCUD1; and SEQ ID NO: 31
for
RNF43.
Derivatives or modified products of antisense oligonucleotides can be used as
antisense oligonucleotides. Examples of such modified products include lower
alkyl
phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-
type,
phosphorothioate modifications and phosphoroamidate modifications.
The term "antisense oligonucleotides" as used herein means, not only those in
which the nucleotides corresponding to those constituting a specified region
of a DNA or
mRNA are entirely complementary, but also those having a mismatch of one or
more
nucleotides, as long as the DNA or mRNA and the antisense oligonucleotide can
specifically hybridize with the nucleotide sequence of SEQ ID NO: 1, 3, or 5.
Such polynucleotides are contained as those having, in the "at least 15
continuous
nucleotide sequence region", a homology of at least 70% or higher, preferably
at 80% or
higher, more preferably 90% or higher, even more preferably 95% or higher. The
algorithm stated herein can be used to determine the homology. Such
polynucleotides are
useful as probes for the isolation or detection of DNA encoding the
polypeptide of the
invention as stated in a later example or as a primer used for amplifications.
The antisense oligonucleotide derivatives of the present invention act upon
cells
producing the polypeptide of the invention by binding to the DNA or mRNA
encoding the
polypeptide, inhibiting its transcription or translation, promoting the
degradation of the


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34
mRNA, and inhibiting the expression of the polypeptide of the invention,
thereby resulting
in the inhibition of the polypeptide's function.
An antisense oligonucleotide derivative of the present invention can be made
into
an external preparation, such as a liniment or a poultice, by mixing with a
suitable base
material which is inactive against the derivatives.
Also, as needed, the derivatives can be formulated into tablets, powders,
granules,
capsules, liposome capsules, injections, solutions, nose-drops and freeze-
drying agents by
adding excipients, isotonic agents, solubilizers, stabilizers, preservatives,
pain-killers, and
such. These can be prepared by following usual methods.
The antisense oligonucleotide derivative is given to the patient by directly
applying
onto the ailing site or by injecting into a blood vessel so that it will reach
the site of ailment.
An antisense-mounting medium can also be used to increase durability and
membrane-permeability. Examples are, liposome, poly-L-lysine, lipid,
cholesterol,
lipofectin or derivatives of these.
The dosage of the antisense oligonucleotide derivative of the present
invention can
be adjusted suitably according to the patient's condition and used in desired
amounts. For
example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be
administered.
The present invention also includes small. interfering RNAs (siRNA) comprising
a
combination of a sense strand nucleic acid and an antisense strand nucleic
acid of the
nucleotide sequence of SEQ ID NO: 1, 3, or 5. More specifically, such siRNA
for
suppressing the expression of RNF43 include those whose sense strand comprises
the
nucleotide sequence of SEQ ID NO: 40, 41, 42, or 43. Alternatively, siRNA for
suppressing the expression of CXADRL1 include those whose sense strand
comprises the
nucleotide sequence of SEQ ID NO: 62, or 63.
The term "siRNA" refers to a double stranded RNA molecule which prevents
translation of a target mRNA. Standard techniques are used for introducing
siRNA into
cells, including those wherein DNA is used as the template to transcribe RNA.
The
siRNA comprises a sense nucleic acid sequence and an anti-sense nucleic acid
sequence of
the polynucleotide encoding human CXADRL1, GCUD1, or RNF43 protein (SEQ ID NO:
1, 3, or 5). The siRNA is constructed such that a single transcript (double
stranded RNA)
has both the sense and complementary antisense sequences from the target gene,
e.g., a
hairpin.
The method is used to alter gene expression of a cell, i.e., up-regulate the
expression of CXADRL1, GCUD1, or RNF43, e.g., as a result of malignant
transformation
of the cells. Binding of the siRNA to CXADRL1, GCUD1, or RNF43 transcript in
the


CA 02488404 2010-12-21

WO 03/104275 PCT/JP03/07006
target cell results in a reduction of protein production by the cell. The
length of the
oligonucleotide is at least 10 nucleotides and may be as long as the naturally
occurring the
transcript. Preferably, the oligonucleotide isl9-25 nucleotides in length.
Most
preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length.
Examples of
5 CXADRLI, GCUD1, or RNF43 siRNA oligonucleotides which inhibit the expression
in
mammalian cells include oligonucleotides containing any of SEQ ID NO: 112-114.
These sequences are target sequence of the following siRNA sequences
respectively.
SEQ ID NO: 112, SEQ ID NOs: 40,41 (RNF43);
SEQ ID NO: 113, SEQ ID NOs: 42, 43 (RNF43); and
10 SEQ ID NO: 114, SEQ ID NOs: 62, 63 (CXADRLI).
The nucleotide sequence of siRNAs may be designed using an siRNA design
computer program available from the Ambion website.
Nucleotide sequences for the siRNA are selected by the computer program based
on the
following protocol:
15 Selection of siRNA Target Sites:
1. Beginning with the AUG start codon of the object transcript, scan
downstream for
AA dinucleotide sequences. Record the occurrence of each AA and the 3'
adjacent
19 nucleotides as potential siRNA target sites. Tuschl, et al. recommend
against
designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near
the
20 start codon (within 75 bases) as these may be richer in-regulatory protein
binding sites.
UTR-binding proteins and/or translation initiation complexes may interfere
with the
binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the human genome database and
eliminate
from consideration any target sequences with significant homology to other
coding
25 sequences. The homology search can be performed using BLAST.

3. Select qualifying target sequences for synthesis. At Ambion, preferably
several
target sequences can be selected along the length of the gene for evaluation.
The antisense oligonucleotide or siRNA of the invention inhibit the expression
of
30 the polypeptide of the invention and is thereby useful for suppressing the
biological
activity of the polypeptide of the invention. Also, expression-inhibitors,
comprising the
antisense oligonucleotide or siRNA of the invention, are useful in the point
that they can
inhibit the biological activity of the polypeptide of the invention.
Therefore, a
composition comprising the antisense oligonucleotide or siRNA of the present
invention is
35 useful in treating a cell proliferative disease such as cancer.
Moreover, the present invention provides a method for diagnosing a cell


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36
proliferative disease using the expression level of the polypeptides of the
present invention
as a diagnostic marker.
This diagnosing method comprises the steps of: (a) detecting the expression
level of
the CX 4DRL1, GCUD1, or RNF43 gene of the present invention; and (b) relating
an
elevation of the expression level to the cell proliferative disease, such as
cancer.
The expression levels of the the CX4DRLI, GCUD1, orRNF43 gene in a particular
specimen can be estimated by quantifying mRNA corresponding to or protein
encoded by
the CX4DRL1, GCUD1, orRNF43 gene. Quantification methods for mRNA are known
to those skilled in the art. For example, the levels of mRNAs corresponding to
the
CXADRLI, GCUD1, or RNF43 gene can be estimated by Northern blotting or RT-PCR.
Since the full-length nucleotide sequences of the CX4DRL1, GCUD1, or RNF43
genes are
shown in SEQ ID NO: 1, 3, or 5, anyone skilled in the art can design the
nucleotide
sequences for probes or primers to quantify the CX4DRL1, GCUD1, orRNF43 gene.
Also the expression level of the CX4DRL1, GCUD1, orRNF43 gene can be
analyzed based on the ctivity or quantity of protein encoded by the gene. A
method for
determining the quantity of the CXADRL1, GCUD1, or RNF43 protein is shown in
bellow.
For example, immunoassay method is useful for the determination of the
proteins in
biological materials. Any biological materials can be used for the
determination of the
protein or it's activity. For example, blood sample is analyzed for estimation
of the
protein encoded by a serum marker. On the other hand, a suitable method can be
selected
for the determination of the activity of a protein encoded by the CXADRLI,
GCUD1, or
RNF43 gene according to the activity of each protein to be analyzed.
Expression levels of the CXADRLI, GCUD1, orRNF43 gene in a specimen (test
sample) are estimated and compared with those in a normal sample. When such a
comparison shows that the expression level of the target gene is higher than
those in the
normal sample, the subject is judged to be affected with a cell proliferative
disease. The
expression level of CX4DRL1, GCUD1, orRNF43 gene in the specimens from the
normal
sample and subject may be determined at the same time. Alternatively, normal
ranges of
the expression levels can be determined by a statistical method based on the
results
obtained by analyzing the expression level of the gene in specimens previously
collected
from a control group. A result obtained by comparing the sample of a subject
is
compared with the normal range; when the result does not fall within the
normal range, the
subject is judged to be affected with the cell proliferative disease. In the
present
invention, the cell proliferative disease to be diagnosed is preferably
cancer. More
preferably, when the expression level of the CX4DRL1, or GCUD1 gene is
estimated and
compared with those in a normal sample, the cell proliferative disease to be
diagnosed is


CA 02488404 2004-12-03
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37
gastric, colorectal, or liver cancer; and when the RNF43 gene is estimated for
its
expression level, then the disease to be diagnosed is colorectal, lung,
gastric, or liver
cancer.
In the present invention, a diagnostic agent for diagnosing cell proliferative
disease,
such as cancer including gastric, colorectal, lung, and liver cancers, is also
provided. The
diagnostic agent of the present invention comprises a compound that binds to a
polynucleotide or a polypeptide of the present invention. Preferably, an
oligonucleotide
that hybridizes to the polynucleotide of the present invention, or an antibody
that binds to
the polypeptide of the present invention may be used as such a compound.
Moreover, the present invention provides a method of screening for a compound
for
treating a cell proliferative disease using the polypeptide of the present
invention. An
embodiment of this screening method comprises the steps of: (a) contacting a
test
compound with a polypeptide of the present invention, (b) detecting the
binding activity
between the polypeptide of the present invention and the test compound, and
(c) selecting a
compound that binds to the polypeptide of the present invention.
The polypeptide of the present invention to be used for screening may be a
recombinant polypeptide or a protein derived from the nature, or a partial
peptide thereof.
Any test compound, for example, cell extracts, cell culture supernatant,
products of
fermenting microorganism, extracts from marine organism, plant extracts,
purified or crude
proteins, peptides, non-peptide compounds, synthetic micromolecular compounds
and
natural compounds, can be used. The polypeptide of the present invention to be
contacted
with a test compound can be, for example, a purified polypeptide, a soluble
protein, a form
bound to a carrier, or a fusion protein fused with other polypeptides.
As a method of screening for proteins, for example, that bind to the
polypeptide of
the present invention using the polypeptide of the present invention, many
methods well
known by a person skilled in the art can be used. Such a screening can be
conducted by,
for example, immunoprecipitation method, specifically, in the following
manner. The
gene encoding the polypeptide of the present invention is expressed in animal
cells and so
on by inserting the gene to an expression vector for foreign genes, such as
pSV2neo,
pcDNA I, and pCD8. The promoter to be used for the expression may be any
promoter
that can be used commonly and include, for example, the SV40 early promoter
(Rigby in
Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141
(1982)),
the EF-1a promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter
(Niwa et al.,
Gene 108: 193-200 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology
152:
684-704 (1987)) the SRa promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)),
the CMV
immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9
(1987)),


CA 02488404 2004-12-03
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38
the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)),
the
Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)), the
HSV TK
promoter, and so on. The introduction of the gene into animal cells to express
a foreign
gene can be performed according to any methods, for example, the
electroporation method
(Chu et al., Nucleic Acids Res 15:1311-26 (1987)), the calcium phosphate
method (Chen
and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata
et al.,
Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4:
1642-3
(1985)), the Lipofectin method (Derijard, B Cell 7: 1025-37 (1994); Lamb et
al., Nature
Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)), and so
on. The
polypeptide of the present invention can be expressed as a fusion protein
comprising a
recognition site (epitope) of a monoclonal antibody by introducing the epitope
of the
monoclonal antibody, whose specificity has been revealed, to the N- or C-
terminus of the
polypeptide of the present invention. A commercially available epitope-
antibody system
can be used (Experimental Medicine 13: 85-90 (1995)). Vectors which can
express a
fusion protein with, for example, (3-galactosidase, maltose binding protein,
glutathione
S-transferase, green florescence protein (GFP) and so on by the use of its
multiple cloning
sites are commercially available.
A fusion protein prepared by introducing only small epitopes consisting of
several
to a dozen amino acids so as not to change the property of the polypeptide of
the present
invention by the fusion is also reported. Epitopes, such as polyhistidine (His-
tag),
influenza aggregate HA, human c-myc, FLAGS Vesicular stomatitis virus
glycoprotein
(VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein
(HSV-tag), E-tag (an epitope on monoclonal phage), and such, and monoclonal
antibodies
recognizing them can be used as the epitope-antibody system for screening
proteins
binding to the polypeptide of the present invention (Experimental Medicine 13:
85-90
(1995)).
In immunoprecipitation, an immune complex is formed by adding these antibodies
to cell lysate prepared using an appropriate detergent. The immune complex
consists of
the polypeptide of the present invention, a polypeptide comprising the binding
ability with
the polypeptide, and an antibody. Immunoprecipitation can be also conducted
using
antibodies against the polypeptide of the present invention, besides using
antibodies
against the above epitopes, which antibodies can be prepared as described
above.
An immune complex can be precipitated, for example by Protein A sepharose or
Protein G sepharose when the antibody is a mouse IgG antibody. If the
polypeptide of the
present invention is prepared as a fusion protein with an epitope, such as
GST, an immune
complex can be formed in the same manner as in the use of the antibody against
the


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39
polypeptide of the present invention, using a substance specifically binding
to these
epitopes, such as glutathione-Sepharose 4B.
Immunoprecipitation can be performed by following or according to, for
example,
the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold
Spring Harbor
Laboratory publications, New York (1988)).
SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the
bound protein can be analyzed by the molecular weight of the protein using
gels with an
appropriate concentration. Since the protein bound to the polypeptide of the
present
invention is difficult to detect by a common staining method, such as
Coomassie staining
or silver staining, the detection sensitivity for the protein can be improved
by culturing
cells in culture medium containing radioactive isotope, 35S-methionine or 35S-
cystein,
labeling proteins in the cells, and detecting the proteins. The target protein
can be
purified directly from the SDS-polyacrylamide gel and its sequence can be
determined,
when the molecular weight of a protein has been revealed.
As a method for screening proteins binding to the polypeptide of the present
invention using the polypeptide, for example, West-Western blotting analysis
(Skolnik et
al., Cell 65: 83-90 (1991)) can be used. Specifically, a protein binding to
the polypeptide
of the present invention can be obtained by preparing a cDNA library from
cells, tissues,
organs (for example, tissues such as testis and ovary for screening proteins
binding to
CXADRL1; testis, ovary, and brain for screening proteins binding to GCUD1; and
fetal
lung, and fetal kidney for those binding to RNF43), or cultured cells expected
to express a
protein binding to the polypeptide of the present invention using a phage
vector (e.g., ZAP),
expressing the protein on LB-agarose, fixing the protein expressed on a
filter, reacting the
purified and labeled polypeptide of the present invention with the above
filter, and
detecting the plaques expressing proteins bound to the polypeptide of the
present invention
according to the label. The polypeptide of the invention may be labeled by
utilizing the
binding between biotin and avidin, or by utilizing an antibody that
specifically binds to the
polypeptide of the present invention, or a peptide or polypeptide (for
example, GST) that is
fused to the polypeptide of the present invention. Methods using radioisotope
or
fluorescence and such may be also used.
Alternatively, in another embodiment of the screening method of the present
invention, a two-hybrid system utilizing cells may be used ("MATCHMAKER
Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit",
"MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector
System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612
(1992)",
"Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").


CA 02488404 2004-12-03
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In the two-hybrid system, the polypeptide of the invention is fused to the
SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA
library is prepared from cells expected to express a protein binding to the
polypeptide of
the invention, such that the library, when expressed, is fused to the VP16 or
GAL4
5 transcriptional activation region. The cDNA library is then introduced into
the above
yeast cells and the cDNA derived from the library is isolated from the
positive clones
detected (when a protein binding to the polypeptide of the invention is
expressed in yeast
cells, the binding of the two activates a reporter gene, making positive
clones detectable).
A protein encoded by the cDNA can be prepared by introducing the cDNA isolated
above
10 to E. coli and expressing the protein.
As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase
gene
and such can be used in addition to the HIS3 gene.
A compound binding to the polypeptide of the present invention can also be
screened using affinity chromatography. For example, the polypeptide of the
invention
15 may be immobilized on a carrier of an affinity column, and a test compound,
containing a
protein capable of binding to the polypeptide of the invention, is applied to
the column. A
test compound herein may be, for example, cell extracts, cell lysates, etc.
After loading
the test compound, the column is washed, and compounds bound to the
polypeptide of the
invention can be prepared.
20 When the test compound is a protein, the amino acid sequence of the
obtained
protein is analyzed, an oligo DNA is synthesized based on the sequence, and
cDNA
libraries are screened using the oligo DNA as a probe to obtain a DNA encoding
the
protein.
Abiosensor using the surface plasmon resonance phenomenon may be used as a
25 mean for detecting or quantifying the bound compound in the present
invention. When
such a biosensor is used, the interaction between the polypeptide of the
invention and a test
compound can be observed real-time as a surface plasmon resonance signal,
using only a
minute amount of polypeptide and without labeling (for example, BlAcore,
Pharmacia).
Therefore, it is possible to evaluate the binding between the polypeptide of
the invention
30 and a test compound using a biosensor such as BlAcore.
The methods of screening for molecules that bind when the immobilized
polypeptide of the present invention is exposed to synthetic chemical
compounds, or
natural substance banks, or a random phage peptide display library, and the
methods of
screening using high-throughput based on combinatorial chemistry techniques
(Wrighton
35 et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996);
Hogan, Nature 384:
17-9 (1996)) to isolate not only proteins but chemical compounds that bind to
the protein


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41
of the present invention (including agonist and antagonist) are well known to
one skilled in
the art.
Alternatively, the screening method of the present invention may comprise the
following steps:
a) contacting a candidate compound with a cell into which a vector comprising
the
transcriptional regulatory region of one or more marker genes and a reporter
gene that is expressed under the control of the transcriptional regulatory
region
has been introduced, wherein the one or more marker genes are selected from
the group consisting of CXADRL1, GCUD1, and RNF43,
b) measuring the activity of said reporter gene; and
c) selecting a compound that reduces the expression level of said reporter
gene as
compared to a control.
Suitable reporter genes and host cells are well known in the art. The reporter
construct required for the screening can be prepared by using the
transcriptional regulatory
region of a marker gene. When the transcriptional regulatory region of a
marker gene has
been known to those skilled in the art, a reporter construct can be prepared
by using the
previous sequence information. When the transcriptional regulatory region of a
marker
gene remains unidentified, a nucleotide segment containing the transcriptional
regulatory
region can be isolated from a genome library based on the nucleotide sequence
information
of the marker gene.
A compound isolated by the screening is a candidate for drugs which promote or
inhibit the activity of the polypeptide of the present invention, for treating
or preventing
diseases attributed to, for example, cell proliferative diseases, such as
cancer. A
compound in which a part of the structure of the compound obtained by the
present
screening method having the activity of binding to the polypeptide of the
present invention
is converted by addition, deletion and/or replacement, is included in the
compounds
obtained by the screening method of the present invention.
In a further embodiment, the present invention provides methods for screening
candidate agents which are potential targets in the treatment of cell
proliferative disease.
As discussed in detail above, by controlling the expression levels of the
CXADRL1,
GCUD1, or RNF43, one can control the onset and progression of either gastric
cancer, or
colorectal, lung, gastric, or liver cancer. Thus, candidate agents, which are
potential
targets in the treatment of cell proliferative disease, can be identified
through screenings
that use the expression levels and activities of CXADRL1, GCUD1, or RNF43 as
indices.
In the context of the present invention, such screening may comprise, for
example, the
following steps:


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42
a) contacting a candidate compound with a cell expressing the CXADRL1, GCUD1,
or RNF43; and
b) selecting a compound that reduces the expression level of CXADRL1, GCUD1,
or RNF43 in comparison with the expression level detected in the absence of
the
test compound.
Cells expressing at least one of the CXADRL1, GCUD1, or RNF43 include, for
example, cell lines established from gastric, colorectal, lung, or liver
cancers; such cells
can be used for the above screening of the present invention. The expression
level can be
estimated by methods well known to one skilled in the art. In the method of
screening, a
compound that reduces the expression level of at least one of CXADRL1, GCUD1,
or
RNF43 can be selected as candidate agents.
In another embodiment of the method for screening a compound for treating a
cell
proliferative disease of the present invention, the method utilizes biological
activity of the
polypeptide of the present invention as an index. Since the CXADRL1, GCUD1,
and
RNF43 proteins of the present invention have the activity of promoting cell
proliferation, a
compound which promotes or inhibits this activity of one of these proteins of
the present
invention can be screened using this activity as an index. This screening
method includes
the steps of: (a) contacting a test compound with the polypeptide of the
present invention;
(b) detecting the biological activity of the polypeptide of step (a); and (c)
selecting a
compound that suppresses the biological activity of the polypeptide in
comparison with the
biological activity detected in the absence of the test compound.
Any polypeptides can be used for screening so long as they comprise the
biological
activity of the CXADRL1, GCUD1, or RNF43 protein. Such biological activity
include
cell-proliferating activity of the human CXADRL1, GCUD1, or RNF43 protein, the
activity of RNF43 to bind to NOTCH2 or STR1N. For example, a human CXADRL1,
GCUD1, or RNF43 protein can be used and polypeptides functionally equivalent
to these
proteins can also be used. Such polypeptides may be expressed endogenously or
exogenously by cells.
Any test compounds, for example, cell extracts, cell culture supernatant,
products
of fermenting microorganism, extracts of marine organism, plant extracts,
purified or crude
proteins, peptides, non-peptide compounds, synthetic micromolecular compounds,
natural
compounds, can be used.
The compound isolated by this screening is a candidate for agonists or
antagonists
of the polypeptide of the present invention. The term "agonist" refers to
molecules that
activate the function of the polypeptide of the present invention by binding
thereto.
Likewise, the term "antagonist" refers to molecules that inhibit the function
of the


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43
polypeptide of the present invention by binding thereto. Moreover, a compound
isolated
by this screening is a candidate for compounds which inhibit the in vivo
interaction of the
polypeptide of the present invention with molecules (including DNAs and
proteins).
When the biological activity to be detected in the present method is cell
proliferation, it can be detected, for example, by preparing cells which
express the
polypeptide of the present invention, culturing the cells in the presence of a
test compound,
and determining the speed of cell proliferation, measuring the cell cycle and
such, as well
as by measuring the colony forming activity as described in the Examples.
The compound isolated by the above screenings is a candidate for drugs which
inhibit the activity of the polypeptide of the present invention and can be
applied to the
treatment of diseases associated with the polypeptide of the present
invention, for example,
cell proliferative diseases including cancer. More particularly, when the
biological
activity of CXADRL1 or GCUD1 protein is used as the index, compounds screened
by the
present method serve as a candidate for drugs for the treatment of gastric,
colorectal, or
liver cancer. On the other hand, when the biological activity of RNF43 protein
is used as
the index, compounds screened by the present method serve as a candidate for
drugs for
the treatment of colorectal, lung, gastric, or liver cancer.
Moreover, compound in which a part of the structure of the compound inhibiting
the activity of CXADRL1, GCUD1, or RNF43 protein is converted by addition,
deletion
and/or replacement are also included in the compounds obtainable by the
screening method
of the present invention.
In a further embodiment of the method for screening a compound for treating a
cell
proliferative disease of the present invention, the method utilizes the
binding ability of
RNF43 to NOTCH2 or STRIN. The RNF43 protein of the present invention was
revealed to associated with NOTCH2 and STRIN. These findings suggest that the
RNF43 protein of the present invention exerts the function of cell
proliferation via its
binding to molecules, such as NOTCH2 and STRIN. Thus, it is expected that the
inhibition of the binding between the RNF43 protein and NOTCH2 or STRIN leads
to the
suppression of cell proliferation, and compounds inhibiting the binding serve
as
pharmaceuticals for treating cell proliferative disease such as cancer.
Preferably, the cell
proliferative disease treated by the compound screened by the present method
is colorectal,
lung, gastric, or liver cancer.
This screening method includes the steps of: (a) contacting a polypeptide of
the
present invention with NOTCH2 or STRIN in the presence of a test compound; (b)
detecting the binding between the polypeptide and NOTCH2 or STRIN; and (c)
selecting
the compound that inhibits the binding between the polypeptide and NOTCH2 or
STRIN.


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The RNF43 polypeptide of the present invention, and NOTCH2 or STRIN to be
used for the screening may be a recombinant polypeptide or a protein derived
from the
nature, or may also be a partial peptide thereof so long as it retains the
binding ability to
each other. The RNF43 polypeptide, NOTCH2 or STRIN to be used in the screening
can
be, for example, a purified polypeptide, a soluble protein, a form bound to a
carrier, or a
fusion protein fused with other polypeptides.
Any test compound, for example, cell extracts, cell culture supernatant,
products of
fermenting microorganism, extracts from marine organism, plant extracts,
purified or crude
proteins, peptides, non-peptide compounds, synthetic micromolecular compounds
and
natural compounds, can be used.
As a method of screening for compounds that inhibit the binding between the
RNF43 protein and NOTCH2 or STRIN, many methods well known by one skilled in
the
art can be used. Such a screening can be carried out as an in vitro assay
system, for
example, in acellular system. More specifically, first, either the RNF43
polypeptide, or
NOTCH2 or STRIN is bound to a support, and the other protein is added together
with a
test sample thereto. Next, the mixture is incubated, washed, and the other
protein bound
to the support is detected and/or measured.
In the same way, a compound interfering the association of CXADRL1 and AIP1
can be isolated by the present invention. It is expected that the inhibition
of the binding
between the CXADRL1 and AIP1 leads to the suppression of cell proliferation,
and
compounds inhibiting the binding serve as pharmaceuticals for treating cell
proliferative
disease such as cancer.
Examples of supports that may be used for binding proteins include insoluble
polysaccharides, such as agarose, cellulose, and dextran; and synthetic
resins, such as
polyacrylamide, polystyrene, and silicon; preferably commercial available
beads and plates
(e.g., multi-well plates, biosensor chip, etc.) prepared from the above
materials may be
used. When using beads, they bay be filled into a column.
The binding of a protein to a support may be conducted according to routine
methods, such as chemical bonding, and physical adsorption. Alternatively, a
protein
may be bound to a support via antibodies specifically recognizing the protein.
Moreover,
binding of a protein to a support can be also conducted by means of avidin and
biotin
binding.
The binding between proteins is carried out in buffer, for example, but are
not
limited to, phosphate buffer and Tris buffer, as long as the buffer does not
inhibit the
binding between the proteins.
In the present invention, a biosensor using the surface plasmon resonance


CA 02488404 2004-12-03
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phenomenon may be used as a mean for detecting or quantifying the bound
protein.
When such a biosensor is used, the interaction between the proteins can be
observed
real-time as a surface plasmon resonance signal, using only a minute amount of
polypeptide and without labeling (for example, BlAcore, Pharmacia). Therefore,
it is
5 possible to evaluate the binding between the RNF43 polypeptide and NOTCH2 or
STRIN
using a biosensor such as BlAcore.
Alternatively, either the RNF43 polypeptide, or NOTCH2 or STRIN, may be
labeled, and the label of the bound protein may be used to detect or measure
the bound
protein. Specifically, after pre-labeling one of the proteins, the labeled
protein is
10 contacted with the other protein in the presence of a test compound, and
then, bound
proteins are detected or measured according to the label after washing.
Labeling substances such as radioisotope (e.g., 3H,14C, 32P, 33P, 35S, 125I,
1311),
enzymes (e.g., alkaline phosphatase, horseradish peroxidase, (3-galactosidase,
P-glucosidase), fluorescent substances (e.g., fluorescein isothiosyanete
(FITC), rhodamine),
15 and biotin/avidin, may be used for the labeling of a protein in the present
method. When
the protein is labeled with radioisotope, the detection or measurement can be
carried out by
liquid scintillation. Alternatively, proteins labeled with enzymes can be
detected or
measured by adding a substrate of the enzyme to detect the enzymatic change of
the
substrate, such as generation of color, with absorptiometer. Further, in case
where a
20 fluorescent substance is used as the label, the bound protein may be
detected or measured
using fluorophotometer.
Furthermore, the binding of the RNF43 polypeptide and NOTCH2 or STRIN can
be also detected or measured using antibodies to the RNF43 polypeptide and
NOTCH2 or
STRIN. For example, after contacting the RNF43 polypeptide immobilized on a
support
25 with a test compound and NOTCH2 or STRIN, the mixture is incubated and
washed, and
detection or measurement can be conducted using an antibody against NOTCH2 or
STRIN.
Alternatively, NOTCH2 or STRIN may be immobilized on a support, and an
antibody
against RNF43 may be used as the antibody.
In case of using an antibody in the present screening, the antibody is
preferably
30 labeled with one of the labeling substances mentioned above, and detected
or measured
based on the labeling substance. Alternatively, the antibody against the RNF43
polypeptide, NOTCH2, or STRIN, may be used as a primary antibody to be
detected with a
secondary antibody that is labeled with a labeling substance. Furthermore, the
antibody
bound to the protein in the screening of the present invention may be detected
or measured
35 using protein G or protein A column.
Alternatively, in another embodiment of the screening method of the present


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46
invention, a two-hybrid system utilizing cells may be used ("MATCHMAKER
Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit",
"MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector
System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612
(1992)",
"Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").
In the two-hybrid system, the RNF43 polypeptide of the invention is fused to
the
SRF-binding region or GAL4-binding region and expressed in yeast cells. The
NOTCH2
or STRIN binding to the RNF43 polypeptide of the invention is fused to the
VP16 or
GAL4 transcriptional activation region and also expressed in the yeast cells
in the
existence of a test compound. When the test compound does not inhibit the
binding
between the RNF43 polypeptide and NOTCH2 or STRIN, the binding of the two
activates
a reporter gene, making positive clones detectable.
As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase
gene
and such can be used besides HIS3 gene.
The compound isolated by the screening is a candidate for drugs which inhibit
the
activity of the RNF43 protein of the present invention and can be applied to
the treatment
of diseases associated with the RNF43 protein, for example, cell proliferative
diseases such
as cancer, more particularly colorectal, lung, gastric, or liver cancer.
Moreover,
compounds in which a part of the structure of the compound inhibiting the
binding
between the RNF43 protein and NOTCH2 or STRIN is converted by addition,
deletion,
substitution and/or insertion are also included in the compounds obtainable by
the
screening method of the present invention.
When administrating the compound isolated by the methods of the invention as a
pharmaceutical for humans and other mammals, such as mice, rats, guinea-pigs,
rabbits,
chicken, cats, dogs, sheep, pigs, cattle, monkeys, baboons, chimpanzees, for
treating a cell
proliferative disease (e.g., cancer) the isolated compound can be directly
administered or
can be formulated into a dosage form using known pharmaceutical preparation
methods.
For example, according to the need, the drugs can be taken orally, as
sugarcoated tablets,
capsules, elixirs and microcapsules, or non-orally, in the form of injections
of sterile
solutions or suspensions with water or any other pharmaceutically acceptable
liquid. For
example, the compounds can be mixed with pharmacologically acceptable carriers
or
medium, specifically, sterilized water, physiological saline, plant-oil,
emulsifiers,
suspending agents, surfactants, stabilizers, flavoring agents, excipients,
vehicles,
preservatives, binders and such, in a unit dose form required for generally
accepted drug
implementation. The amount of active ingredients in these preparations makes a
suitable
dosage within the indicated range acquirable.


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Examples of additives that can be mixed to tablets and capsules are, binders
such as
gelatin, corn starch, tragacanth gum and arabic gum; excipients such as
crystalline
cellulose; swelling agents such as corn starch, gelatin and alginic acid;
lubricants such as
magnesium stearate; sweeteners such as sucrose, lactose or saccharin;
flavoring agents
such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage
form is a
capsule, a liquid carrier, such as oil, can also be further included in the
above ingredients.
Sterile composites for injections can be formulated following normal drug
implementations
using vehicles such as distilled water used for injections.
Physiological saline, glucose, and other isotonic liquids including adjuvants,
such
as D-sorbitol, D-mannnose, D-mannitol, and sodium chloride, can be used as
aqueous
solutions for injections. These can be used in conjunction with suitable
solubilizers, such
as alcohol, specifically ethanol, polyalcohols such as propylene glycol and
polyethylene
glycol, non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used
in
conjunction with benzyl benzoate or benzyl alcohol as a solubilizers and may
be
formulated with a buffer, such as phosphate buffer and sodium acetate buffer;
a pain-killer,
such as procaine hydrochloride; a stabilizer, such as benzyl alcohol, phenol;
and an
anti-oxidant. The prepared injection may be filled into a suitable ampule.
Methods well known to one skilled in the art may be used to administer the
inventive pharmaceutical compound to patients, for example as intraarterial,
intravenous,
percutaneous injections and also as intranasal, transbronchial, intramuscular
or oral
administrations. The dosage and method of administration vary according to the
body-weight and age of a patient and the administration method; however, one
skilled in
the art can routinely select them. If said compound is encodable by a DNA, the
DNA can
be inserted into a vector for gene therapy and the vector administered to
perform the
therapy. The dosage and method of administration vary according to the body-
weight,
age, and symptoms of a patient but one skilled in the art can select them
suitably.
For example, although there are some differences according to the symptoms,
the
dose of a compound that binds with the polypeptide of the present invention
and regulates
its activity is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg
to about 50
mg per day and more preferably about 1.0 mg to about 20 mg per day, when
administered
orally to a normal adult (weight 60 kg).
When administering parenterally, in the form of an injection to a normal adult
(weight 60 kg), although there are some differences according to the patient,
target organ,
symptoms and method of administration, it is convenient to intravenously
inject a dose of
about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per
day and


CA 02488404 2004-12-03
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48
more preferably about 0.1 to about 10 mg per day. Also, in the case of other
animals too,
it is possible to administer an amount converted to 60kgs of body-weight.
Moreover, the present invention provides a method for treating or preventing a
cell
proliferative disease, such as cancer, using an antibody against the
polypeptide of the
present invention. According to the method, a pharmaceutically effective
amount of an
antibody against the polypeptide of the present invention is administered.
Since the
expression of the CXADRLI, GCUD1, and RNF43 protein are up-regulated in cancer
cells,
and the suppression of the expression of these proteins leads to the decrease
in cell
proliferating activity, it is expected that cell proliferative diseases can be
treated or
prevented by binding the antibody and these proteins. Thus, an antibody
against the
polypeptide of the present invention are administered at a dosage sufficient
to reduce the
activity of the protein of the present invention, which is in the range of 0.1
to about 250
mg/kg per day. The dose range for adult humans is generally from about 5 mg to
about
17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about
100 mg to
about 3 g/day.
Alternatively, an antibody binding to a cell surface marker specific for tumor
cells can be used as a tool for drug delivery. For example, the antibody
conjugated with a
cytotoxic agent is administered at a dosage sufficient to injure tumor cells.
The present invention also relates to a method of inducing anti-tumor immunity
comprising the step of administering CXADRL1, GCUD1, or RNF43 protein or an
immunologically active fragment thereof, or a polynucleotide encoding the
protein or
fragments thereof. The CXADRL1, GCUD1, or RNF43 protein or the immunologically
active fragments thereof are useful as vaccines against cell proliferative
diseases. In some
cases the proteins or fragments thereof may be administered in a form bound to
the T cell
recepor (TCR) or presented by an antigen presenting cell (APC), such as
macrophage,
dendritic cell (DC), or B-cells. Due to the strong antigen presenting ability
of DC, the use
of DC is most preferable among the APCs.
In the present invention, vaccine against cell proliferative disease refers to
a
substance that has the function to induce anti-tumor immunity upon inoculation
into
animals. According to the present invention, polypeptides comprising the amino
acid
sequence of SEQ ID NO: 80, 97, or 108 were suggested to be HLA-A24 or HLA-
A*0201
restricted epitopes peptides that may induce potent and specific immune
response against
colorectal, lung, gastric, or liver cancer cells expressing RNF43. According
to the present
invention, polypeptides comprising the amino acid sequence of SEQ ID NO:124
was
suggested to be HLA-A*0201 restricted epitopes peptides that may induce potent
and
specific immune response against colorectal, gastric, or liver cancer cells
expressing


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CXADRLI. According to the present invention, polypeptides comprising the amino
acid
sequence of SEQ ID NO: 164 was suggested to be HLA-A*0201 restricted epitopes
peptides that may induce potent and specific immune response against
colorectal, gastric,
or liver cancer cells expressing GCUD1. Thus, the present invention also
encompasses
method of inducing anti-tumor immunity using polypeptides comprising the amino
acid
sequence of SEQ ID NO: 80, 97, 108, 124 or 164. In general, anti-tumor
immunity
includes immune responses such as follows:
- induction of cytotoxic lymphocytes against tumors,
- induction of antibodies that recognize tumors, and
- induction of anti-tumor cytokine production.
Therefore, when a certain protein induces any one of these immune responses
upon inoculation into an animal, the protein is decided to have anti-tumor
immunity
inducing effect. The induction of the anti-tumor immunity by a protein can be
detected
by observing in vivo or in vitro the response of the immune system in the host
against the
protein.
For example, a method for detecting the induction of cytotoxic T lymphocytes
is
well known. A foreign substance that enters the living body is presented to T
cells and B
cells by the action of antigen presenting cells (APCs). T cells that respond
to the antigen
presented by APC in antigen specific manner differentiate into cytotoxic T
cells (or
cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen, and then
proliferate
(this is referred to as activation of T cells). Therefore, CTL induction by a
certain peptide
can be evaluated by presenting the peptide to T cell by APC, and detecting the
induction of
CTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ T cells,
macrophages, eosinophils, and NK cells. Since CD4+ T cells and CD8+ T cells
are also
important in anti-tumor immunity, the anti-tumor immunity inducing action of
the peptide
can be evaluated using the activation effect of these cells as indicators.
A method for evaluating the inducing action of CTL using dendritic cells (DCs)
as
APC is well known in the art. DC is a representative APC having the strongest
CTL
inducing action among APCs. In this method, the test polypeptide is initially
contacted
with DC, and then this DC is contacted with T cells. Detection of T cells
having
cytotoxic effects against the cells of interest after the contact with DC
shows that the test
polypeptide has an activity of inducing the cytotoxic T cells. Activity of CTL
against
tumors can be detected, for example, using the lysis of 51Cr-labeled tumor
cells as the
indicator. Alternatively, the method of evaluating the degree of tumor cell
damage using
3H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as the
indicator is
also well known.


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Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as
the APC. The induction of CTL is reported that the it can be enhanced by
culturing
PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be
induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH)
and
5 IL-7.
The test polypeptides confirmed to possess CTL inducing activity by these
methods are polypeptides having DC activation effect and subsequent CTL
inducing
activity. Therefore, polypeptides that induce CTL against tumor cells are
useful as
vaccines against tumors. Furthermore, APC that acquired the ability to induce
CTL
10 against tumors by contacting with the polypeptides are useful as vaccines
against tumors.
Furthermore, CTL that acquired cytotoxicity due to presentation of the
polypeptide
antigens by APC can be also used as vaccines against tumors. Such therapeutic
methods
for tumors using anti-tumor immunity due to APC and CTL are referred to as
cellular
immunotherapy.
15 Generally, when using a polypeptide for cellular immunotherapy, efficiency
of the
CTL-induction is known to increase by combining a plurality of polypeptides
having
different structures and contacting them with DC. Therefore, when stimulating
DC with
protein fragments, it is advantageous to use a mixture of multiple types of
fragments.
Alternatively, the induction of anti-tumor immunity by a polypeptide can be
20 confirmed by observing the induction of antibody production against tumors.
For
example, when antibodies against a polypeptide are induced in a laboratory
animal
immunized with the polypeptide, and when growth of tumor cells is suppressed
by those
antibodies, the polypeptide can be determined to have an ability to induce
anti-tumor
immunity.
25 Anti-tumor immunity is induced by administering the vaccine of this
invention,
and the induction of anti-tumor immunity enables treatment and prevention of
cell
proliferating diseases, such as gastric, colorectal, lung, and liver cancers.
Therapy against
cancer or prevention of the onset of cancer includes any of the steps, such as
inhibition of
the growth of cancerous cells, involution of cancer, and suppression of
occurrence of
30 cancer. Decrease in mortality of individuals having cancer, decrease of
tumor markers in
the blood, alleviation of detectable symptoms accompanying cancer, and such
are also
included in the therapy or prevention of cancer. Such therapeutic and
preventive effects
are preferably statistically significant. For example, in observation, at a
significance level
of 5% or less, wherein the therapeutic or preventive effect of a vaccine
against cell
35 proliferative diseases is compared to a control without vaccine
administration. For
example, Student's t-test, the Mann-Whitney U-test, or ANOVA may be used for
statistical


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51
analyses.
The above-mentioned protein having immunological activity or a vector encoding
the protein may be combined with an adjuvant. An adjuvant refers to a compound
that
enhances the immune response against the protein when administered together
(or
successively) with the protein having immunological activity. Examples of
adjuvants
include cholera toxin, salmonella toxin, alum, and such, but are not limited
thereto.
Furthermore, the vaccine of this invention may be combined appropriately with
a
pharmaceutically acceptable carrier. Examples of such carriers are sterilized
water,
physiological saline, phosphate buffer, culture fluid, and such. Furthermore,
the vaccine
may contain as necessary, stabilizers, suspensions, preservatives,
surfactants, and such.
The vaccine is administered systemically or locally. Vaccine administration
may be
performed by single administration, or boosted by multiple administrations.
When using APC or CTL as the vaccine of this invention, tumors can be treated
or
prevented, for example, by the ex vivo method. More specifically, PBMCs of the
subject
receiving treatment or prevention are collected, the cells are contacted with
the polypeptide
ex vivo, and following the induction of APC or CTL, the cells may be
administered to the
subject. APC can be also induced by introducing a vector encoding the
polypeptide into
PBMCs ex vivo. APC or CTL induced in vitro can be cloned prior to
administration. By
cloning and growing cells having high activity of damaging target cells,
cellular
immunotherapy can be performed more effectively. Furthermore, APC and CTL
isolated
in this manner may be used for cellular immunotherapy not only against
individuals from
whom the cells are derived, but also against similar types of tumors from
other individuals.
Furthermore, a pharmaceutical composition for treating or preventing a cell
proliferative disease, such as cancer, comprising a pharmaceutically effective
amount of
the polypeptide of the present invention is provided. The pharmaceutical
composition
may be used for raising anti tumor immunity. The normal expression of CXADRL1,
restricted to testis and ovary; normal expression of GCUD1 is restricted to
testis, ovary,
and brain; and normal expression of RNF43 is restricted to fetus, more
specifically to fetal
lung and kidney. Therefore, suppression of these genes may not adversely
affect other
organs. Thus, the CXADRL1 and GCUD1 polypeptides are preferable for treating
cell
proliferative disease, especially gastric, colorectal, or liver cancer; and
RNF43 polypeptide
is also preferable for treating cell proliferative disease, especially
colorectal, lung, gastric,
and liver cancers. Furthermore, since peptide fragments of RNF43 comprising
the amino
acid sequences of SEQ ID NO: 80, 97, and 108, respectively, were revealed to
induce
immune response against RNF43, polypeptides comprising the amino acid sequence
of
SEQ ID NO: 80, 97, or 108 are preferable examples of polypeptides that can be
used in a


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52
pharmaceutical composition for treating or preventing cell proliferative
disease, especially
colorectal, lung, gastric, and liver cancers. Furthermore, since peptide
fragments of
CXADRL1 comprising the amino acid sequences of SEQ ID NO: 124, respectively,
were
revealed to induce immune response against CXADRL1, polypeptides comprising
the
amino acid sequence of SEQ ID NO: 124 is preferable examples of polypeptide
that can be
used in a pharmaceutical composition for treating or preventing cell
proliferative disease,
especially colorectal, lung, gastric, and liver cancers. Furthermore, since
peptide
fragments of GCUD1 comprising the amino acid sequences of SEQ ID NO: 164,
respectively, was revealed to induce immune response against GCUD1,
polypeptides
comprising the amino acid sequence of SEQ ID NO: 164 is preferable examples of
polypeptides that can be used in a pharmaceutical composition for treating or
preventing
cell proliferative disease, especially colorectal, lung, gastric, and liver
cancers. In the
present invention, the polypeptide or fragment thereof is administered at a
dosage
sufficient to induce anti-tumor immunity, which is in the range of 0.1 mg to
10 mg,
preferably 0.3mg to 5mg, more preferably 0.8mg to 1.5 mg. The administrations
are
repeated. For example, 1mg of the peptide or fragment thereof may be
administered 4
times in every two weeks for inducing the anti-tumor immunity.
The following examples are presented to illustrate the present invention and
to
assist one of ordinary skill in making and using the same. The examples are
not intended
in any way to otherwise limit the scope of the invention.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, suitable
methods and materials are described below. Any patents, patent applications,
and
publications cited herein are incorporated by reference.

Best Mode for Carrying out the Invention
The present invention is illustrated in details by following Examples, but is
not
restricted to these Examples.
1. Materials and Methods
(1) Patients and tissue specimens
All gastric and colorectal cancer tissues, as well as corresponding non-
cancerous
tissues were obtained with informed consent from surgical specimens of
patients who
underwent surgery.
(2) Genome-wide cDNA microarray


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53
In-house genome-wide cDNA microarray comprising 23040 genes were used in
this study. DNase I treated total RNA extracted from microdissected tissue was
amplified
with Ampliscribe T7 Transcription Kit (Epicentre Technologies) and labeled
during reverse
transcription with Cy-dye (Amersham) (RNA from non-cancerous tissue with Cy5
and
RNA from tumor with Cy3). Hybridization, washing, and detection were carried
out as
described previously (Ono et al., Cancer Res. 60: 5007-11 (2000)), and
fluorescence
intensity of Cy5 and Cy3 for each target spot was measured using Array Vision
software
(Amersham Pharmacia). After subtraction of background signal, duplicate values
were
averaged for each spot. Then, all fluorescence intensities on a slide were
normalized to
adjust the mean Cy5 and Cy3 intensity of 52 housekeeping genes for each slide.
Genes
with intensities below 25,000 fluorescence units for both Cy3 and Cy5 were
excluded from
further investigation, and those with Cy3/Cy5 signal ratios > 2.0 were
selected for further
evaluation.
(3) Cell lines
Human embryonic kidney 293 (HEK293) were obtained TaKaRa. COST cell, NIH3T3
cell, human cervical cancer cell line HeLa, human gastric cancer cell lines
MKN-1 and
MKN-28, human hepatoma cell line Alexander, and human colon cancer cell lines,
LoVo,
HCT116, DLD-1 and SW480, were obtained from the American Type Culture
Collection
(ATCC, Rockville, MD). Human hepatoma cell line SNU475 and human colon cancer
cell lines, SNUC4 and SNUC5, were obtained from the Korea cell-line bank. All
cells
were grown in monolayers in appropriate media: Dulbecco's modified Eagle's
medium for
COST, NIH3T3, HEK293, and Alexander; RPMI1640 for MKN-1, MKN-28, SNU475,
SNUC4, DLD-1 and SNUC5; McCoy's 5A medium for HCT116; Leibovitz's L-15 for
SW480; HAM's F-12 for LoVo; and Eagle's minimum essential medium for HeLa
(Life
Technologies, Grand Island, NY). All media were supplemented with 10% fetal
bovine
serum and 1% antibiotic/antimycotic solution (Sigma). A human gastric cancer
cell lines
St-4 was kindly provided by Dr. Tsuruo in Cancer Institure in Japan. St-4
cells were
grown in monolayers in RPMI1640 supplemented with 10% fetal bovine serum and
1%
antibiotic/antimycotic solution (Sigma).
T2 cells (HLA-A*0201) and EHM (HLA-A3/3), human B-lymphoblastoid cell lines,
were
generous gifts from Prof. Shiku (Univ. Mie). HT29 (colon carcinoma cell line;
HLA-A24/01), WiDR (colon carcinoma cell line; HLA-A24/01), and HCT116 (colon
carcinoma cell line; HLA-A02/01), DLD-1 (colon carcinoma cell line; HLA-
A24/01),
SNU475 (hepatocellular carcinoma cell line; HLA-A*0201), MKN45 (gastric cancer
cell
line; HLA-A2 negative), MKN74 (gastric cancer cell line; HLA-A2 negative) were
also
purchased from ATCC. TISI cells (HLA-A24/24) were generous gifts from Takara
Shuzo


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Co, Ltd. (Otsu, Japan). RT-PCR examinations revealed strong CXADRL1 expression
in
SNU475 and MKN74. RT-PCR examinations revealed strong GCUD1 expression in
SNU475 and MKN45.

(4) RNA preparation and RT-PCR
Total RNA was extracted with Qiagen RNeasy kit (Qiagen) or Trizol reagent
(Life
Technologies) according to the manufacturers' protocols. Ten-microgram
aliquots of total
RNA were reversely transcribed for single-stranded cDNAs using poly dT12.18
primer
(Amersham Pharmacia Biotech) with Superscript II reverse transcriptase (Life
Technologies). Each single-stranded cDNA preparation was diluted for
subsequent PCR
amplification by standard RT-PCR experiments carried out in 20~u1 volumes of
PCR buffer
(TaKaRa). Amplification was conducted under following conditions: denaturing
for 4
min at 94 C, followed by 20 (for GAPDH), 35 (for CXADRL1), 30 (for GCUD1), 30
(for
RNF43) cycles of 94 C for 30 s, 56 C for 30 s, and 72 C for 45 s, in
GeneAmp PCR
system 9700 (Perkin-Elmer, Foster City, CA). Primer sequences were; for GAPDH:
forward, 5'-ACAACAGCCTCAAGATCATCAG (SEQ ID NO: 7) and reverse,
5'-GGTCCACCACTGACACGTTG (SEQ ID NO: 8); for CXADRL1: forward,
5'-AGCTGAGACAT1TGTTCTCTTG (SEQ ID NO: 9) and reverse: 5'-TATAAACCAG
CTGAGTCCAGAG (SEQ ID NO: 10); for GCUD1 forward:
5'-TTCCCGATATCAACATCTACCAG (SEQ ID NO: 11) reverse:
5'-AGTGTGTGACCTCAATAAGGCAT (SEQ ID NO: 12), for RNF43 forward;
5'-CAGGCTTTGGACGCACAGGACTGGTAC-3' (SEQ ID NO: 13) and reverse;
5'-CTTTGTGATCATCCTGGCTTCGGTGCT 3' (SEQ ID NO: 14).
(5) Northern-blot analysis
Human multiple-tissue blots (Clontech, Palo Alto, CA) were hybridized with
32P-labeled PCR products of CXADRL1, GCUD1, or RNF43. Pre-hybridization,
hybridization and washing were performed according to the supplier's
recommendations.
The blots were autoradiographed with intensifying screens at -80 C for 24 to
72 h.
(6) 5' rapid amplification of cDNA ends (5' RACE)
5' RACE experiments were carried out using Marathon cDNA amplification kit
(Clontech) according to the manufacturer's instructions. For the amplification
of the 5'
part of CXADRL1, gene-specific reverse primers
(5'-GGTTGAGATTTAAGTTCTCAAA-3' (SEQ ID NO: 15)) and the AP-1 primer
supplied with the kit were used. The cDNA template was synthesized from human
testis
mRNA(Clontech). The PCR products were cloned using TA cloning kit (Invitrogen)
and
their sequences were determined with ABI PRISM 3700 DNA sequencer (Applied


CA 02488404 2004-12-03
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Biosystems).
(7) Construction of plasmids expressing CXADRL1, GCUD1, and FLJ20315
The entire coding regions of CXADRL1, GCUD1, and RNF43 were amplified by
RT-PCR using gene specific primer sets; for CXADRL1,
5 5'-AGTTAAGCTTGCCGGGATGACTTCTCAGCGTTCCCCTCTGG-3' (SEQ ID NO:
16) and 5'-ATCTCGAGTACCAAGGACCCGGCCCGACTCTG-3' (SEQ ID NO: 17), for
GCUD15'-GCGGATCCAGGATGGCTGCTGCAGCTCCTCCAAG-3' (SEQ ID NO: 18)
and 5'-TAGAATTCTTAAAGAACTTAATCTCCGTGTCAACAC-3' (SEQ ID NO: 19),
for RNF43, 5'-TGCAGATCTGCAGCTGGTAGCATGAGTGGTG-3' (SEQ ID NO: 20)
10 and 5'-GAGGAGCTGTGTGAACAGGCTGTGTGAGATGT 3' (SEQ ID NO: 21). The
PCR products were cloned into appropriate cloning site of either pcDNA3.1
(Invitrogen),
or pcDNA3.1myc/His (Invitrogen) vector.
(8) Immunoblotting
Cells transfected with pcDNA3.1myc/His-CXADRL1,
15 pcDNA3.1myc/His-GCUD1, pcDNA3.1myc/His-RNF43 or pcDNA3.1myc/His-LacZ
were washed twice with PBS and harvested in lysis buffer (150 mM NaCl, 1%
Triton
X-100, 50 mM Tris-HCl pH 7.4, 1mM DTT, and 1X complete Protease Inhibitor
Cocktail
(Boehringer)). Following homogenization, the cells were centrifuged at
10,000xg for 30
min, the supernatant were standardized for protein concentration by the
Bradford assay
20 (Bio-Rad). Proteins were separated by 10% SDS-PAGE and immunoblotted with
mouse
anti-myc (SANTA CRUZ) antibody. HRP-conjugated goat anti-mouse IgG (Amersham)
served as the secondary antibody for the ECL Detection System (Amersham).
(9) Immunohistochemical staining
Cells transfected with pcDNA3.1myc/His-CXADRL1,
25 pcDNA3.1myc/His-GCUD1, pcDNA3.lmyc/His-RNF43 or pcDNA3.1myc/His-LacZ
were fixed with PBS containing 4% paraformaldehyde for 15 min, then made
permeable
with PBS containing 0.1% Triton X-100 for 2.5 min at RT. Subsequently the
cells were
covered with 2% BSA in PBS for 24 h at 4 C to block non-specific
hybridization. Mouse
anti-myc monoclonal antibody (Sigma) at 1:1000 dilution was used as the
primary
30 antibody, and the reaction was visualized after incubation with Rhodamine-
conjugated
anti-mouse secondary antibody (Leinco and ICN). Nuclei were counter-stained
with
4',6'-diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescent images
were
obtained under an ECLIPSE E800 microscope.
(10) Colony Formation assay
35 Cells transfected with plasmids expressing each gene or control plasmids
were
incubated with an appropriate concentration of geneticin for 10 to 21 days.
The cells


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56
were fixed with 100% methanol and stained by Giemsa solution. All experiments
were
carried out in duplicate.
(11) Establishment of cells over-expressing CXADRL1, or RNF43
NIH3T3, COST, and LoVo cells transfected with either
pcDNA3.1myc/His-CXADRLI, pcDNA3.1myc/His-RNF43, pcDNA3.lmyc/His-LacZ or
control plasmids, respectively, were maintained in media containing
appropriate
concentration of geneticin. Two weeks after the transfection, surviving single
colonies
were selected, and expression of each gene was examined by semi-quantitative
RT-PCR.
(12) Examination on the effect of anti-sense oligonucleotides on cell growth
Cells plated onto 10-cm dishes (2X105 cells/dish) were transfected either with
plasmid, or synthetic S-oligonucleotides of CXADRL1, GCUD1, or RNF43 using
LIPOFECTIN Reagent (GIBCO BRL). Then the cells were cultured with the addition
of
an appropriate concentration of geneticin for six to twelve days. The cells
were then
fixed with 100% methanol and stained by Giemsa solution. Sequences of the
S-oligonucleotides were as follows:
CXADRL1-S4, 5'-TCTGCACGGTGAGTAG-3' (SEQ ID NO: 22);
CXADRL1-AS4, 5'-CTACTCACCGTGCAGA-3' (SEQ ID NO: 23);
CXADRL1-S5, 5'-TTCTGTAGGTGTTGCA-3' SEQ ID NO: 24);
CXADRL1-AS5, 5'-TGCAACACCTACAGAA-3' (SEQ ID NO: 25);
GCUD1-S5, 5'-CTTVFCAGGATGGCTG-3' (SEQ ID NO: 26);
GCUD1-AS5, 5'-CAGCCATCCTGAAAAG-3' (SEQ ID NO: 27);
GCUD1-S8, 5'-AGGTTGAGGTAAGCCG-3' (SEQ ID NO: 28);
GCUD1-AS8, 5'-CGGCTTACCTCAACCT 3' (SEQ ID NO: 29);
RNF43-S1, 5'-TGGTAGCATGAGTGGT 3' (SEQ ID NO; 30); and
RNF43-AS1, 5'-ACCACTCATGCTACCA-3' (SEQ ID NO: 31).
(13)(4,5-dimethylthiazol-2-yl -2 5-diphenyltetrazolium bromide (MTT) assay
Cells plated at a density of 5X105 cells/100 mm dish were transfected in
triplicate
with sense or antisense S-oligonucleotides designated to suppress the
expression of
CXADRL1, GCUD1 or RNF43. Seventy-two hours after transfection, the medium was
replaced with fresh medium containing 500 g/ml of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) and
the plates
were incubated for four hours at 37 C. Subsequently, the cells were lysed by
the addition
of 1 ml of 0.01 N HCl/10%SDS and the absorbance of lysates was measured with
ELISA
plate reader at a test wavelength of 570 nm (reference, 630 nm). The cell
viability was
represented by the absorbance compared to that of control cells.
(14) Construction of psiHlBX3.0


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Since H1RNA gene was reported to be transcribed by RNA polymerase III, which
produce short transcripts with uridines at the 3' end, a genomic fragment of
H1RNA gene
containing its promoter region was amplified by PCR using a set of primers
[5'-TGGTAGCCAAGTGCAGGTTATA-3' (SEQ ID NO: 32), and 5'-
CCAAAGGGTTTCTGCAGMCA-3' (SEQ ID NO: 33)] and human placental DNA as a
template. The products were purified and cloned into pCR2.0 plasmid vector
using TA
cloning kit (Invitrogen) according to the supplier's protocol. The BamHI and
XhoI
fragment containing the H1RNA gene was purified and cloned into pcDNA3.1(+)
plasmid
at the nucleotide position from 1257 to 56, which plasmid was amplified by PCR
with a set
of primers, 5'-TGCGGATCCAGAGCAGATTGTACTGAGAGT 3' (SEQ ID NO: 34) and
5'-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3' (SEQ ID NO: 35), and then
digested with BamHI and Xhol. The ligated DNA was used as a template for PCR
with
primers,
5'- TTTAAGCTTGAAGACCATTTYFGG C-3'
(SEQ ID NO: 36) and 5'-TTTAAGCTTGAAGACATGGGAAAGAGTGGTCTCA-3'
(SEQ ID NO: 37). The product was digested with HindIII, and subsequently self-
ligated
to produce psiH1BX3.0 vector plasmid. As the control, psiHlBX-EGFP was
prepared by
cloning double-stranded oligonucleotides of
5'- CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC
-3' (SEQ ID NO: 38) and
5'- AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC
-3' (SEQ ID NO: 39) into the BbsI site of the psiH1BX vector.
(15) Examination on the gene silencing effect of RNF43-, or CXADRL1-siRNAs
A plasmid expressing either RNF43-siRNA or CXADRL1-siRNA was prepared
by cloning of double-stranded oligonucleotides into psiH1BX3.0 vector.
Oligonucleotides used as RNF43 siRNAs were:
5'- TCCCGTCACCGGATCCAACTCAGTTCAAGAGACTGAGTTGGATCCGGTGA
C-3' (SEQ ID NO: 40) and
5' -AAAAGTCACCGGATCCAACTCAGTCTCTTGAACTGAGTTGGATCCGGTGAC-
3' (SEQ ID NO: 41) as siRNA16-4;
5'- TCCCGCTATTGCACAGAACGCAGTTCAAGAGACTGCGTTCTGTGCAATAGC-
3' (SEQ ID NO: 42) and
5'-AAAAGCTATTGCACAGAACGCAGTCTCTTGAACTGCGTTCTGTGCAATAGC-3'
(SEQ ID NO: 43) as siRNA1834;
5'- TCCCCAGAAAGCTGTTATCAGAGTTCAAGAGACTCTGATAACAGCTTTCTG-
3' (SEQ ID NO: 44) and


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58
5' -AAAACAGAAAGCTGTTATCAGAGTCTCTTGAACTCTGATAACAGCTTTCTG-3'
(SEQ ID NO: 45) as siRNA1;
5'- TCCCTGAGCCACCTCCAATCCACTTCAAGAGAGTGGATTGGAGGTGGCTCA-
3' (SEQ ID NO: 46) and
5'-AAAATGAGCCACCTCCAATCCACTCTCTTGAAGTGGATTGGAGGTGGCTCA-
3' (SEQ ID NO: 47) as siRNA14;
5'- TCCCCTGCACGGACATCAGCCTATTCAAGAGATAGGCTGATGTCCGTGCAG-
3' (SEQ ID NO: 48) and
5' -AAAACTGCACGGACATCAGCCTATCTCTTGAATAGGCTGATGTCCGTGCAG-3'
(SEQ ID NO: 49) as siRNA15. Oligonucleotides used as CXADRL1- siRNAs we
re: 5' -TCCCGTGTCAGAGAGCCCTGGGATTCAAGAGATCCCAGGGCTCTCTGAC
AC-3' (SEQ ID NO: 50) and 5'-AAAAGTGTCAGAGAGCCCTGGGATCTCTTGAA
TCCCAGGGCTCTCTGACAC-3' (SEQ ID NO: 51) as siRNA#1; 5'-TCCCCCTCAA
TGTCATTTGGATGTTCAAGAGACATCCAAATGCAATTGAGG-3' (SEQ ID NO: 5
2) and 5'-AAAACCTCAATGTCATTTGGATGTCTCTTGAACATCCAAATGCAATTG
AGG-3' (SEQ ID NO: 53) as siRNA#2; 5'-TCCCTGTCATTTGGATGGTCACTTTC
AAGAGAAGTGACCATCCAAATGACA-3' (SEQ ID NO: 54) and 5'-AAAATGTCA
TTTGGATGGTCACTTCTCTTGAAAGTGACCATCCAAATGACA-3' (SEQ ID NO:
55) as siRNA#3; 5'-TCCCTGCCAACCAACCTGAACAGTTCAAGAGACTGTTCAG
GTTGGTTGGCA-3' (SEQ ID NO: 56) and 5'-AAAATGCCAACCAACCTGAACAG
TCTCTTGAACTGTTCAGGTTGGTTGGCA-3' (SEQ ID NO: 57) as siRNA#4; 5'-T
CCCCCAACCTGAACAGGTCATCTTCAAGAGAGATGACCTGTTCAGGTTGG-3' (S
EQ ID NO: 58) and 5'-AAAACCAACCTGAACAGGTCATCTCTCTTGAAGATGAC
CTGTTCAGGTTGG-3' (SEQ ID NO: 59) as siRNA#5; 5'-TCCCCCTGAACAGGTC
ATCCTGTTTCAAGAGAACAGGATGACCTGTTCAGG-3' (SEQ ID NO: 60) and 5'
-AAAACCTGAACAGGTCATCCTGTTCTCTTGAAACAGGATGACCTGTTCAGG-3'
(SEQ ID NO: 61) as siRNA#6; and 5'-TCCCCAGGTCATCCTGTATCAGGTTCAAG
AGACCTGATACAGGATGACCTG-3' (SEQ ID NO: 62) and 5'-AAAACAGGTCAT
CCTGTATCAGGTCTCTTGAACCTGATACAGGATGACCTG-3' (SEQ ID NO: 63) a
s CXADRL-siRNA#7. psiHlBX-RNF43, psiHlBX-CXADRL1, or psiHlBX-mock pl
asmids were transfected into SNUC4 or St-4 cells using FuGENE6 reagent (Roche)
according to the supplier's recommendations. Total RNA was extracted from the
cells 48 hours after the transfection.
(16) Construction of recombinant amino- and carboxyl-terminal regions of RNF43
protein
The amino- and carboxyl-terminal regions of RATF43 was amplified by RT-PCR
using following sets of primers: 5'-GAAGATCTGCAGCGGTGGAGTCTGAAAG-3'


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(SEQ ID NO: 64) and 5'-GGAAfTCGGACTGGGAAAATGAATCTCCCTC-3' (SEQ ID
NO: 65) for the amino-terminal region; and
5'-GGAGATCTCCTGATCAGCAAGTCACC-3' (SEQ ID NO: 66) and
5'-GGAATTCCACAGCCTGTTCACACAGCTCCTC-3' (SEQ ID NO: 67) for the
carboxyl-terminal region. The products were digested with BamHI-EcoRI and
cloned
into the BamHI-EcoRI site of pET-43.la(+) vector (Novagen). The plasmids were
transfected into E. coli BL21trxB(DE3)pLysS cells (Stratagene) . Recombinant
RNF43
protein was extracted from cells cultured at 25 C for 16 h after the addition
of 0.2 mM
IPTCL
(17) Yeast two-hybrid experiment
Yeast two-hybrid assay was performed using MATCHMAKER GAL4
Two-Hybrid System 3 (Clontech) according to the manufacturer's protocols. The
entire
coding sequence of RNF43 was cloned into the EcoR I-BarnH I site of pAS2-1
vector as a
bait for screening human-testis cDNA library (Clontech). To confirm the
interaction in
yeast, pAS2-RNF43 was used as bait vector, pACT2-NOTCH2 and pACT2-STRIN as
prey
vector.
We cloned the cytoplasmic region of CXADRL1 into the EcoRI site of pAS2-1
vector as a bait for screening a human testis cDNA library (Clontech). To
confirm
interaction in yeast, we used pAS2-CXADRL1 for bait vector, and pACT2-AIP1 for
prey
vector.

(18) Preparation of CXADRL specific antibody
Anti-CXADRL antisera was prepared by immunization with synthetic
polypeptides of CXADRL1 encompassing codons from 235 to 276 for Ab-1, from 493
to
537 for Ab-2, or from 70 to 111 for Ab-3. Sera were purified using recombinant
His-tagged CXADRL1 protein prepared in E. coli transfected with pET CXADRL
plasmid.
Protein extracted from cells expressing Flag-tagged CXADRL1 was further
separated by
10% SDS-PAGE and immunoblotted with either anti-CXADRL1 sera or anti-Flag
antibody.
HRP-conjugated goat anti-rabbit IgG or HRP-conjugated sheep anti-mouse IgG
antibody
served as the secondary antibody, respectively, for ECL Detection System
(Amersham
Pharmacia Biotech, Piscataway, NJ). Immunoblotting with anti-CXADRL antisera
showed a 50 kD band of FLAG-tagged CXADRL1, which pattern was identical to
that
detected with anti-FLAG antibody.
(19) Preparation of recombinant GCUD1 protein
To generate an antibody specific against GCUD1, recombinant GCUD1 protein
was prepared. The entire coding region of GCUDI was amplified by RT-PCR with a
set


CA 02488404 2004-12-03
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of primers, 5'-GCGGATCCAGGATGGCTGCAGCTCCTCCAAG-3' (SEQ ID NO: 68)
and 5'-CTGAATTCACTTAAAGAACTTAATCTCCGTGTCAACAC-3' (SEQ ID NO: 69).
The product was purified, digested with BamH1 and EcoRl, and cloned into an
appropriate
cloning site of pGEX6P-2. The resulting plasmid was dubbed pGEX-GCUD1.
5 pGEX-GCUD1 plasmid was transformed into E. coli DH1OB. The production of the
recombinant protein was induced by the addition of IPTQ and the protein was
purified
with Glutathione SepharoseTm 4B (Amersham Pharmacia) according to the
manufacturers'
protocols.
(20) Preparation of GCUD1 specific antibody
10 Polyclonal antibody against GCUD1 was purified from sera. Proteins from
cells
transfected with plasmids expressing Flag-tagged GCUD1 were separated by 10%
SDS-PAGE and immunoblotted with anti-GCUD1 or anti-Flag antibody.
HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA)
or
HRP-conjugated anti-Flag antibody served as the secondary antibody,
respectively, for
15 ECL Detection System (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunoblotting
with the anti-GCUD1 antibody showed a 47 kD band of FLAG-tagged GCUD1, which
pattern was identical to that detected with the anti-FLAG antibody.
(21) Statistical analysis
The data were subjected to analysis of variance (ANOVA) and the Scheffe's F
test.
20 (22) Preparation of peptides
9mer and 10mer peptides of RNF43, CXADRLI or GCUD1 that bind to HLA-A24
or HIA-A*0201 molecule were predicted with the aide of binding prediction soft
(http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker_comboform). These
peptides were
synthesized by Mimotopes, San Diego, LA according to the standard solid phase
synthesis
25 method and purified by reversed phase HPLC. The purity (>90%) and the
identity of the
peptides were determined by analytical HPLC and mass spectrometry analysis,
respectively.
Peptides were dissolved in dimethylsulfoxide (DMSO) at 20 mg/ml and stored at -
80 C.
(23) In vitro CTL Induction
Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells
30 (APCs) to induce CTL responses against peptides presented on HLA. DCs were
generated in vitro as described elsewhere (Nukaya et al., Int J Cancer 80: 92-
7 (1999); Tsai
et al., J Immunol 158: 1796-802 (1997)). Specifically, peripheral blood
mononuclear
cells (PBMCs) were isolated from a healthy volunteer with HIA-A*0201 or HLA-
A24
using Ficoll-Plaque (Pharmacia) solution, and monocyte fraction of PBMCs were
35 separated by adherence to a plastic tissue culture flask (Becton
Dickinson). This
monocyte fraction was cultured for seven days in AIM-V medium (Invitrogen)
containing


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2% heat-inactivated autologous serum (AS), 1000 U/ml of GM-CSF (provided by
Kirin
Brewery Company), and 1000 U/ml of IL-4 (Genzyme) to obtain DCs fraction. The
20
g/ml of candidate peptides were pulsed onto this DC enriched cell population
in the
presence of 3 g/ml of (32-microglobulin for 4 hat 20 C in AIM-V. These
peptide-pulsed
antigen presenting cells were then irradiated (5500 rads) and mixed at a 1:20
ratio with
autologous CD8+ T cells, obtained by positive selection with Dynabeads M-450
CD8
(Dynal) and Detachabead (Dynal). These cultures were set up in 48-well plates
(Coming); each well contained 1.5x104 peptide-pulsed antigen presenting cells,
3x105
CD8+ T cells and 10 ng/ml of IL-7 (Genzyme) in 0.5 ml of AIM-V with 2% AS.
Three
days later, these cultures were supplemented with IL-2 (CHIRON) to a final
concentration
of 20 IU/ml. On day 7 and 14, the T cells were further restimulated with the
autologous
peptide-pulsed antigen presenting cells which were prepared each time in the
same manner
as described above. Lymphoid cells in the culture on day 21 were harvested and
tested
for cytotoxicity against peptide-pulsed TISI or T2 cells.
(24) CTL Expansion
Cultured lymphoid cells with proved significant cytotoxicity against
peptide-pulsed TISI or T2 were further expanded in culture using a method
similar to that
described by Riddell, et al. (Walter et al., N Engl J Med 333:1038-1044, 1995;
Riddel et al.,
Nature Med. 2:216-223, 1996 ). 5 x 104 of lymphoid cells were resuspended in
25 ml of
AIM-V supplemented with 5% AS containing 25 x 106 irradiated (3300 rads) PBMC,
5 x
106 irradiated (8000 rads) EHM cells, and 40 ng/ml of anti-CD3 monoclonal
antibody
(Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2 were
added to the
cultures. The cultures comprised fresh AIM-V supplemented with 5% AS and 30
IU/ml
of IL-2 on days 5, 8 and 11.
(25) Establishment of CTL clones
Some of the lymphoid cells with potent cytotoxicity were used to obtain CTL
clones. The cell suspensions were diluted to density of 0.3, 1, and 3
CTLs/lymphoid cells
per well in 96 round-bottom microtiter plate (Nalge Nunc International). These
cells
were cultured in 150 1/well of AIM-Vsupplemented with 5%AS containing 7x104
cells/well of allogenic PBMCs,1x104 cells/well of EHM, 30ng/ml of anti-CD3
antibody,
and 125 U/ml of IL-2. 10 days later, 50 l /well of IL-2 was added to the
medium to a
final concentration of 125 U/ml. Cytotoxic activity of cultured CTLS was
tested on day
14, and CTL clones were expanded using the same method as described above.
(26) CytotoxicityAssay
Target cells were labeled with 100 Ci of Na251CrO4 (Perkin Elmer Life
Sciences) for 1 h
at 37 C in a CO2 incubator. When peptide-pulsed targets were used, target
cells were


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incubated with the addition of 20 g/ml of the peptide for 16 h at 37 C before
the labeling
with Na251CrO4. Target cells were rinsed and mixed with effectors at a final
volume of
0.2 ml in round-bottom microtiter plates. The plates were centrifuged (4
minutes at 800 x
g) to increase cell-to-cell contact and placed in a CO2 incubator at 37 C.
After 4 h of
incubation, 0.1 ml of the supernatant was collected from each well and the
radioactivity
was determined with a gamma counter. In case of evaluating cytotoxicity
against target
cells that endogenously express RNF43 or CXADRLI or GCUD1, the cytolytic
activity was
tested in the presence of a 30-fold excess of unlabeled K562 cells to reduced
any
non-specific lysis due to NK-like effectors. Antigen specificity was confirmed
by the cold
target inhibition assay, which utilized unlabeled TISI or T2 cells that were
pulsed with
peptide (20 U g/ml for 16 hrs at 37 C) to compete for the recognition of 51Cr-
labeled
HT29 or SNU475 cells. The MHC restriction was examined by blocking assay,
measuring
the inhibition of the cytotoxicity by anti HLA-class I (W6/32) antibody and
anti HLA-class
II antibody, anti CD4 antibody and anti CD8 antibody (DAKO).
The percentage of specific cytotoxicity was determined by calculating the
percentage of specific 51Cr - release by the following formula: {(cpm of the
test sample
release - cpm of the spontaneous release)/(cpm of the maximum release - cpm of
the
spontaneous release)} X 100. Spontaneous release was determined by incubating
the
target cells alone in the absence of effectors, and the maximum release was
obtained by
incubating the targets with 1N HCI. All determinants were done in duplicate,
and the
standard errors of the means were consistently below 10% of the value of the
mean.

2. Results
(1) Identification of two novel human genes, CXADRL1 and GCUD1, commonly
up-regulated in gastric cancers
By means of a genome-wide cDNA microarray containing 23040 genes,
expression profiles of 20 gastric cancers were compared with corresponding non-
cancerous
mucosae. Among commonly up-regulated genes detected in the microarray
analysis, a
gene with an in-house accession number of A5928 corresponding to an EST,
Hs.6658 of
UniGene cluster, was found to be over-expressed
in a range between 4.09 and 48.60 (Figure la). Since an open reading frame of
this gene
encoded a protein approximately 37% identical to that of CXADR (Coxsackie and
adenovirus receptor), this gene was dubbed CXADRLI (Coxsackie and adenovirus
receptor
like 1). CXADRLI was also up-regulated in 6 of 6 colorectal cancer cases and
12 out of 20
HCC cases. Furthermore, a gene with an in-house accession number of C8121,
corresponding to KIA.A0913 gene product (Hs.75137) of UniGene cluster was also
focused


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due to its significantly enhanced expression in nine of twelve gastric cancer
tissues
compared with the corresponding non-cancerous gastric mucosae by microarray
(Figure
1b). This gene with the in-house accession number C8121 was dubbed GCUD1
(up-regulated in gastric cancer). GCUD1 was also up-regulated in 5 of 6
colorectal cancer
cases, 1 out of 6 HCC cases, 1 out of 14 lung cancer(squeamous cell carcinoma)
cases, 1
out of 13 testicular seminomas cases. To clarify the results of the cDNA
microarray,
expression of these transcripts in gastric cancers was examined by semi-
quantitative
RT-PCR to confirm an increased expression of CXADRL1 in all of the 10 tumors
(Figure
1c) and elevated expression of GCUD1 in seven of nine cancers (Figure 1d).
(2) Isolation and structure of a novel gene CXADRLI
Multiple-tissue northern-blot analysis using a PCR product of CXADRLI as a
probe revealed the expression of a 3.5-kb transcript in testis and ovary
(Figure 2a). Since
A5928 was smaller than the gene detected on the Northern blot, 5'RACE
experiments were
carried out to determine the entire coding sequence of the CXADRLI gene. The
putative
full-length cDNA consisted of 3423 nucleotides, with an open reading frame of
1296
nucleotides (SEQ ID NO: 1) encoding a 431-amino-acid protein (SEQ ID NO: 2)
(GenBank Accession number: AB071618). The first ATG was flanked by a sequence
(CCCGGGATGA) (SEQ ID NO: 70) that was consistent with the consensus sequence
for
the initiation of translation in eukaryotes, with an in-frame stop codon
upstream. Using
the BLAST program to search for homologies in the NCBI (the National Center
for
Biotechnology Information) databases, a genomic sequence with the GenBank
accession
number AC068984 was identified, which sequence had been assigned to
chromosomal
band 3q13. Comparison of the cDNA and the genomic sequence revealed that
CXADRL1 consisted of 7 exons (Figure 2b).
A search for protein motifs using the Simple Modular Architecture Research
Tool
revealed that the predicted protein contained two immunogloblin domains
(codons 29-
124 and 158-232) and a transmernbrane domain (codons 246-268), suggesting that
CXADRL I might belong to the immunogloblin super family.

(3) Effect of CXADRL1 on cell growth
A colony-formation assay was performed by transfecting NIH3T3 cells with a
plasmid expressing CXADRL1(pcDNA3.1myc/His-CXADRL1). Cells transfected with
pcDNA3.lmyc/His-CXADRLI produced markedly more colonies than mock-transfected
cells (Figure 3a). To further investigate this growth-promoting effect of
CXADRL1,
NIH3T3 cells that stably expressed exogenous CXADRLI were established (Figure.
3b).
The growth rate of NIH3T3-CXADRL1 cells was significantly higher than that of
parental


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NIH3T3 cells in culture media containing 10% FBS (Figure 3c).
(4) Suppression of CXADRL1 expression in human gastric cancer cells by
antisense
S-oligonucleotides
Six pairs of control and antisense S-oligonucleotides corresponding to CXADRLI
were transfected into MKN-1 gastric cancer cells, which had shown the highest
level of
CXADRL1 expression among the examined six gastric cancer cell lines. Six days
after
transfection, viability of transfected cells was measured by MTT assay. Viable
cells
transfected with antisense S-oligonucleotides (CXADRL1-AS4 or -AS5) were
markedly
fewer than those transfected with control S-oligonucleotides (CXADRL1-S4 or -
S5)
(Figure 4). Consistent results were obtained in three independent experiments.
(5) Construction of plasmids expressing CXADRL1 siRNAs and their effect on the
growth
of gastric cancer cells
Plasmids expressing various CXADRLI-siRNA were constructed and examined
for their effect on CXADRL1 expression. Among the constructed siRNAs,
psiHlBX-CXADRL7 significantly suppressed the expression of CXADRL1 in St-4
cells
(Figure 5A). To test whether the suppression of CXADRL1 may result in growth
suppression of colon cancer cells, St-4 cells were transfected with psiHlBX-
CXADRL7 or
mock vector. The number of viable cells transfected with psiH1BX-CXADRL7 was
fewer than the number of viable control cells (Figure 5B and 5C).
(6) Preparation of anti-CXADRL1 antibody
To examine the expression and explore the function of CXADRL1, anti-sera
against CXADRL1 was prepared. Immunoblotting with anti-CXADRL1 detected a 50
kD band of FLAG-tagged CXADRL1, which was almost identical by size to that
detected
with anti-FLAG antibody (Figure 6).
(7) Identification of a CXADRL1-interacting protein by yeast two-hybrid
screening
system.
To clarify the function of CXADRL1, we searched for CXADRL1-interacting
proteins using yeast two-hybrid screening system. Among the positive clones
identified,
C-terminal region of nuclear AIP1 (atrophin interacting protein 1) interacted
with
CXADRL1 by simultaneous transformation using pAS2.1-CXADRL1 and pACT2-AIP1
(Figure 7) in the yeast cells. The positive clones contained codons between
808 and 1008,
indicating that responsible region for the interaction in AIP1 is within this
region.
(8) Prediction of candidate peptides derived from CXADRL1
Table 1 show the candidate peptides (SEQ ID NOs: 115-154) in order of high
binding affinity. Forty peptides in total were selected and examined as
described below.


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Tablel Prediction of candidate peptides derived from CXADRLI
HLA-A*02019 mer HLA-A*0201 10 mer
Rank sequence Score Position Rank sequence Score Position
1 YLVrEKLDNT 1314.7 176 1 YLWEkLDNTL 3344 176
2 LLLLSLHGV 1006.2 11 2 LINLnVIWMV 280.45 52
3 1 NLM/1 ww 49.262 53 3 ALSSgLYQCV 104.33 207
4 WIV TPLSNA 37.961 59 4 ALININVIWM 62.845 51
5 CLVNNLPDI 23.995 120 5 ILLCsSEEGI 32.155 162
6 SLHGVAASL 21.362 15 6 VLPCtFTTSA 32.093 41
7 VIII FG AL 18.975 252 7 LLLSIHGVAA 31.249 12
8 LI NLNUI M 14.69 52 8 SIYAnGTHLV 30.603 356
9 AVLPCTFTT 13.993 40 9 QLSDtGTYQC 20.369 111
10 ALSSGLYQC 11.426 207 10 GLYQcVASNA 15.898 211
11 VNBRSNGSV 11.101 384 11 PLLLISLHGV 13.022 10
12 SI FI NNTQL 10.868 104 12 IQVArGQPAV 11.988 32
13 KvI-IRNTDSV 10.437 327 13 FINNtQLSDT 10.841 106
14 RI GbVPVW 9.563 413 14 LVPGqHKTLV 10.346 364
15 NI GVrGLTV 9.563 132 15 NLPDiGGRNI 8.555 124
16 SI YANGTHL 9.399 356 16 VLVPpSAPHC 8.446 140
17 LLCSSEEG 8.691 163 17 AVIIiFCIAL 7.103 251
18 LLSLHGVAA 8.446 13 18 VIIIfCIALI 5.609 252
19 I I FG ALI L 7.575 254 19 ILGAfFYWRS 5.416 261
20 TM'ATNVSI 7.535 97 20 GLTVIVPPSA 4.968 137
(9) Stimulation of the T cells and establishment of CTL clones using the
candidate peptides
5 Lymphoid cells were cultured using these candidate peptides derived from
CXADRLI in the manner described in "Materials and Methods". Resulting lymphoid
cells showing detectable cytotoxic activity were expanded, and CTL clone was
established.
CTL clone was propagated from the CTL lines described above using limiting
dilution
methods. CTL clone induced with CXADRL1-207 (ALSSGLYQC) (SEQ ID NO: 124)
10 showed the higher cytotoxic activities against the target pulsed with
peptides when
compared with those against targets not pulsed with any peptides. Cytotoxic
activity of
this CTL clone was shown in Figure 8. This CTL clone had very potent cytotoxic
activity
against the peptide-pulsed target without showing any cytotoxic activity
against the target
not pulsed with any peptides.
15 (10) Cytotoxic activity against tumor cell lines endogenously expressing
CXADRLI as a
target
The CTL clones raised against predicted peptides were examined for their
ability to
recognize and kill the tumor cells endogenously expressing CXADRL1. Figure 9
shows
the results of CTL Clone 75 raised against CXADRL1-207 (ALSSGLYQC) (SEQ ID NO:
20 124). CTL Clone 75 showed potent cytotoxic activity against SNU475 which
expresses


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CXADRL1 and HLA-A*0201, however it did not show against MKN74 which expressed
CXADRLI but not HLA-A*0201, and did not show against SNU-C4 which expressed
HLA-A* 0201 but not CXADRL1.
(11) Specificity of the established CTLs
Cold target inhibition assay was also performed to confirm the specificity of
CXADRL1-207 CTL Clone. SNU475 cells labeled by 51Cr were used as a hot target,
while T2 cells pulsed with CXADRL1-207 (SEQ ID NO: 124) were used without 51Cr
labeling as a cold target. Specific cell lysis against SNU475 cells was
significantly
inhibited, when T2 pulsed with CXADRL1-207 (SEQ ID NO: 124) was added in the
assay
at various ratios (Figure 10). Theses results were indicated as a percentage
of specific
lysis at the E/T ratio of 20.
Regarding CXADRL1-207 CTL clone, to examine the characteristics of these
CTL clones, antibodies against HLA-Class I, HLA-Class II, CD4 and CD8 were
tested for
their capacity to inhibit the cytotoxic activity. The cytotoxicity of CTL
clone against
SNLJ475 cells was significantly inhibited when anti-HLA-Class I antibody and
anti-CD8
antibody were used (Figure 11), indicating that the CTL clone recognize the
CXADRL1
derived peptide in a HLA-Class I and CD8 manner.
(12) Expression and characterization of novel human gene GCUD1
Multi-tissue northern blot analysis using GUCD1 cDNA as a probe showed a
5.0-kb transcript expressed specifically in testis, ovary, and brain (Figure
12). Although
the nucleotide sequence of KIAA0913 (GenBank Accession Number: XM-014766),
corresponding to GCUD1, consisted of 4987 nucleotides, RT-PCR experiments
using testis,
ovary and cancer tissues revealed a transcript that consisted of 4987
nucleotides containing
an open reading frame of 1245 nucleotides (SEQ ID NO: 3) (GenBank Accession
Number:
AB071705). Furthermore, the genomic sequence corresponding to GUCD1 was
searched
in genomic databases to find a draft sequence assigned to chromosomal band
7p14
(GenBank Accession Number: NT 007819). Comparison between the cDNA sequence
and the genomic sequence revealed that the GUCD1 gene consisted of 8 exons.
(13) Subcellular localization of GCUD1
The entire coding region corresponding to GCUD1 was cloned into
pCDNA3.1myc/His vector and the construct was transiently transfected into COST
cell.
Immunocytochemical staining of the COST cell revealed that the tagged-GCUD1
protein
was present in the cytoplasm (Figure 13).
(14) Effect of GCUD1 on cell growth
To analyze the effect of GCUD1 on cell growth, a colony-formation assay was
conducted by transfecting NIH3T3 cells with a plasmid expressing GCUD1


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(pcDNA3.1myc/His-GCUD1). Compared with a control plasmid
(pcDNA3.1myc/His-LacZ), pcDNA3.lmyc/His-GCUD1 induced markedly more colonies
in NIH3T3 cells (Figure 14). This result was confirmed by three independent
experiments.
(15) Growth suppression of gastric cancer cells by antisense S-
oligonucleotides designated
to reduce expression of GCUD1
To test whether the suppression of GCUD1 may result in cell death of gastric
cancer cells, various antisense S-oligonucleotides designed to suppress the
expression of
GCUD1 were synthesized. Six days after transfection of the respective
antisense
S-oligonucleotides, viability of transfected cells was measured by MTT assay.
Viable
cells transfected with antisense S-oligonucleotides (GCUD1-AS5 or -AS8) were
markedly
fewer than those transfected with control S-oligonucleotides (GCUD1-S5 or -S8)
in
MKN-28 cells (Figure 15). This result was confirmed by three independent
experiments.
Similar result was observed with MKN-1 cells.
(16) Preparation of anti-GCUD1 antibody
To examine the expression and explore the function of GCUD1, anti-sera against
GCUD1 was prepared. Recombinant protein of GCUD1 was extracted and purified
from
bacterial cells expressing GST-GCUD1 fusion protein (Figure 16). The
recombinant
protein was used for immunization of three rabbits. Immunoblotting with anti-
GCDU1
sera but not pre-immune sera showed a 47 kD band of FLAG-tagged GCUD1, which
was
almost identical by size to that detected with anti-FLAG antibody (Figure 17).
(17) Prediction of candidate peptides derived from GCUD1
Table 2 (GCUD1) shows the candidate peptides (SEQ ID NOs: 155-194) in order of
high binding affinity. Forty peptides in total were selected and examined as
described
below.


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Table. 2 Prediction of candidate peptides derived from GCUD1
HLA-A*02019 mer HLA-A*020110 mer
Rank sequence Score Position Rank sequence Score Position
1 SIFKPFIFV 369.77 303 1 FIFVdDVKLV 374.37 308
2 WLWGAEMGA 189.68 75 2 LIVDrDEAWV 366.61 158
3 IMISRPAWL 144.26 68 3 FLTTaSGVSV 319.94 272
4 LLGMDLVRL 83.527 107 4 TMLEIEKQGL 234.05 371
5 FIFVDDVKL 49.993 308 5 ALLGmDLVRL 181.79 106
6 VCIDSEFFL 31.006 265 6 AIMIsRPAWL 59.775 67
7 KPFIFVDDV 25.18 306 7 GVCIdSEFFL 59.628 264
8 IVDRDEAWV 22.761 159 8 KLVPkTQSPC 17.388 315
9 TLRDKASGV 21.672 257 9 FNFSeVFSPV 14.682 220
10 KMDAEHPEL 21.6 196 10 YISIdQVPRT 10.841 56
11 ALDVIVSLL 19.653 126 11 GEGEfNFSEV 10.535 216
12 YAQSQGWWT 19.639 207 12 WAAEkVTEGV 8.927 175
13 KLRSTMLEL 13.07 367 13 VLPQnRSSPC 8.446 281
14 YLIVDRDEA 11.198 157 14 AAAPpSYCFV 7.97 2
AAPPpYCFV 7.97 3 15 TMMNtLRDKA 6.505 253
16 GMDLVRLGL 6.171 109 16 EVGDIFYDCV 5.227 397
15 17 KVTEGVRCI 6.026 179 17 AEMGaNEHGV 5.004 79
18 CIDSEFFLT 4.517 266 18 GLVVfGKNSA 4.968 20
19 TVQTMMNTL 4.299 250 19 QLSLtTKMDA 4.968 190
EMGANEHGV 3.767 -CO 20 RSIFkPFIFV 4.745 302
(18) Stimulation of the T cells and establishment of CTL clones using the
candidate
20 peptides
Lymphoid cells were cultured using these candidate peptides derived from GCUD1
in the manner described in "Materials and Methods". Resulting lymphoid cells
showing
detectable cytotoxic activity were expanded, and CTL clones were established.
CTL
clones were propagated from the CTL lines described above using limiting
dilution
methods. CTL clones induced with GCUD1-196 (KMDAEHPEL) (SEQ ID NO: 164)
and GCUD1-272 (FLTTASGVSV) (SEQ ID NO: 177) showed the higher cytotoxic
activities against the target pulsed with peptides when compared with those
against targets
not pulsed with any peptides. Cytotoxic activity of these CTL clones was shown
in
Figure 18. Each CTL clone had very potent cytotoxic activity against the
peptide-pulsed
target without showing any cytotoxic activity against the target not pulsed
with any
peptides.
(19) Cytotoxic activity against tumor cell lines endogenously expressing GCUD1
as a
target
The CTL clones raised against predicted peptides were examined for their
ability to
recognize and kill the tumor cells endogenously expressing GCUD1. Figure 19
shows the
results of CTL Clone 23 raised against GCUD1-196 (SEQ ID NO: 164). CTL Clone
23


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showed potent cytotoxic activity against SNU475 which expresses GCUD1 and
HLA-A*0201, however it did not show against MKN45 which expressed GCUD1 but
not
HLA-A*0201.
(20) Specificity of the established CTLs
Cold target inhibition assay was also performed to confirm the specificity of
GCUD1-196 CTL Clone. SNU475 cells labeled by 51Cr were used as a hot target,
while
T2 cells pulsed with GCUDl-196 were used without 51Cr labeling as a cold
target.
Specific cell lysis against SNU475 cells was significantly inhibited, when T2
pulsed with
GCUD1-196 (SEQ ID NO: 164) was added in the assay at various ratios (Figure
20).
Theses results were indicated as a percentage of specific lysis at the E/T
ratio of 20.
Regarding GCUDI-196 (SEQ ID NO: 164) CTL clone, to examine the
characteristics of these CTL clones, antibodies against HLA-Class I, HLA-Class
II, CD4
and CD8 were tested for their capacity to inhibit the cytotoxic activity. The
cytotoxicity of
CTL clone against SNU475 cells was significantly inhibited when anti-HLA-Class
I
antibody and anti-CD8 antibody were used (Figure 21), indicating that the CTL
clone
recognize the GCUD1 derived peptide in a HLA-Class I and CD8 manner.
(21) Identification of gene FLJ20315 commonly up-regulated in human colon
cancer
Expression profiles of 11 colon cancer tissues were compared with non-
cancerous
mucosal tissues of the colon corresponding thereto using the cDNA microarray
containing
23040 genes. According to this analysis, expression levels of a number of
genes that
were frequently elevated in cancer tissues compared to corresponding non-
cancerous
tissues. Among them, a gene with an in-house accession number of B4469
corresponding
to an EST (FLJ20315), Hs.18457 in UniGene cluster
was up-regulated in the cancer tissue compared
to the corresponding non-cancerous mucosae at a magnification range between
1.44 and
11.22 (Figure 22a). FLJ20315 was also up-regulated in 6 out of 18 gastric
cancer cases,
12 out of 20 HCC cases, 11 out of 22 lung cancer(adenocarcinoma) cases, 2 out
of 2
testicular seminomas cases and 3 out of 9 prostate cancer cases. To clarify
the results of
the microarray, the expression of these transcripts in additional colon cancer
samples were
examined by semi-quantitative RT-PCR to confirm the increase of FLJ20315
expression in
15 of the 18 tumors (Figure 22b).
(22) Expression and Structure of RNF43
Additional homology searches of the sequence of FLf20315 in public databases
using BLAST program in National Center for Biotechnology Information
identified ESTs including XM097063, BF817142 and a genomic sequence with a
GenBank Accession Number NT-010651 assigned to


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chromosomal band 17pter-p13.1. As a result, an assembled sequence of 5345
nucleotides
containing an open reading frame of 2352 nucleotides (SEQ ID NO: 5) encoding a
783-amino-acid protein (SEQ ID NO: 6) (GenBank Accession Number: AB081837) was
obtained. The gene was dubbed RNF43 (Ring finger protein 43). The first ATG
was
5 flanked by a sequence (AGCATGC) that agreed with the consensus sequence for
initiation
of translation in eukaryotes, and by an in-frame stop codon upstream.
Comparison of the
cDNA and the genomic sequence revealed that this gene consisted of 11 exons.
Northern-blot analysis using human adult Multiple-Tissue northern-blots with a
PCR
product of RNF43 as a probe failed to detect any band (data not shown).
However, a 5.2
10 kb-transcript was detected to be expressed in fetal lung and fetal kidney
using a human
fetal tissue northern-blot with the same PCR product as a probe (Figure 23a).
A search
for protein motifs with the Simple Modular Architecture Research Tool (SMART,
revealed that the predicted protein contained a Ring finger motif (codons 272-
312)(Figure 23b).
15 (23) Subcellular localization of myc-tagged RNF43 protein
To investigate the subcellular localization of RNF43 protein, a plasmid
expressing
myc-tagged RNF43 protein (pDNAmyc/His-RNF43) was transiently transfected into
COST cells. Western blot analysis using extracts from the cells and anti-myc
antibody
revealed a 85.5-KDa band corresponding to the tagged protein (Figure 24a).
Subsequent
20 immunohistochemical staining of the cells with the same antibody indicated
the protein to
be mainly present in the nucleus (Figure 24b). Similar subcellular
localization of RNF43
protein was observed in SW480 human colon cancer cells.
(24) Effect of RNF43 on cell growth
A colony-formation assay was conducted by transfecting NIH3T3 cells with a
25 plasmid expressing RNF43 (pcDNA-RNF43). Cells transfected with pcDNA-RNF43
produced significantly more number of colonies than control cells (Figure
25a).
Increased activity of colony formation by RNF43 was also shown in SW480 cells
wherein
the endogenous expression of RNF43 was very low (data not shown). To further
investigate this growth-promoting effect, COST cells that stably express
exogenous RNF43
30 (COS7-RNF43) were established (Figure 25b). The'growth rate of COS7-RNF43
cells
was significantly higher than that of COS7-mock cells in culture media
containing 10%
FBS (Figure 25c).
(25) Growth suppression of colon cancer cells by antisense S-oligonucleotides
designated
to reduce expression of RNF43
35 To test whether the suppression of RNF43 expression may result in growth
retardation and/or cell death of colon cancer cells, five pairs of control and
antisense


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S-oligonucleotides corresponding to RNF43 were synthesized and transfected
into LoVo
colon cancer cells, which shown a higher level of RNF43 expression among the
examined
11 colon cancer cell lines. Among the five antisense S-oligonucleotides, RNF43-
AS1
significantly suppressed the expression of RNF43 compared to control S-
oligonucleotides
(RNF43-S1)12 hours after transfection (Figure 26a). Six days after
transfection, number
of surviving cells transfected with RNF43-AS1 was significantly fewer than
those
transfected with RNF43-S1 suggesting that the suppression of RNF43 expression
reduced
growth and/or survival of transfected cells (Figure 26b). Consistent results
were obtained
in three independent experiments. -
(26) Construction of plasmids expressing RNF43 siRNAs and their effect on
growth of
colon cancer cells
In mammalian cells, small interfering RNA (siRNA) composed of 20 or 21-mer
double-stranded RNA (dsRNA) with 19 complementary nucleotides and 3' terminal
complementary dimmers of thymidine or uridine, has been recently shown to have
a gene
specific gene silencing effect without inducing global changes in gene
expression.
Therefore, plasmids expressing various RNF43-siRNAs were constructed to
examine their
effect on RNF43 expression. Among the various RNF43-siRNAs, psiHlBX-RNF16-4
and psiH1BX-RNF1834 significantly suppressed the expression of RNF43 in SNUC4
cells
(Figure 27A). To test whether the suppression of RNF43 results in growth
suppression of
colon cancer cells, SNUC4 cells were transfected with psiHlBX-RNF16-4,
psiH1BX-RNF1834 or mock vector. In line with the data of antisense S-
oligonucleotides,
the number of viable cells transfected with psiHlBX-RNF16-4 or psiHlBX-RNF1834
was
fewer than the number of viable control cells (Figure 27B, 27C).
(27) Secretion of Flag-tagged RNF43 protein in culture media of COS7 cells
with
exogenous Flag-tagged RNF43 protein
Since a search for protein motifs with amino acid sequence of RNF43 using
Simple Modular Architecture Research Tool
predicted a signal peptide and a ring finger domain, secretion of the RNF43
protein was
examined. Plasmid expressing Flag-tagged RNF43 (pFLAG-RNF43) or Myc-tagged
RNF43 (pcDNA3.1-Myc/His-RNF43), or mock vector was transfected into COST
cells,
and the cells were cultured in media supplemented with 0.5 % of bovine calf
serum for 48
hours . Western blot analysis with anti-Flag antibody or anti-Myc antibody
detected
secreted Flag-tagged RNF43 or Myc-tagged protein in the media containing cells
transfected with pFLAG-RNF43 or pcDNA3.1-Myc/His-RNF43, respectively, but not
in
the media containing cells with mock vector (Figure 28A and 28B).
(28) Effect of cultured media of cells transfected with_pFLAG-RNF43 on N1H3T3
cells


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Since exogenous expression of RNF43 conferred growth promoting effect on
NIH3T3 cells, secreted Flag-tagged RNF43 was examined whether it also has a
proliferative effect on NIH3T3 cells. NIH3T3 cells were cultured without the
change of
media, or with conditioned media of cells transfected with mock-transfected
cells, or those
with pFlag-RNF43. As expected, cells transfected with either pFlag-RNF43 or
pcDNA3.1-Myc/His-RNF43 cultured in conditioned media showed a significantly
higher
growth rate compared to non-treated cells or mock-vector transfected cells
cultured in
conditioned media (Figure 29A and 29B). These data suggest that RNF43 may
exert its
growth promoting effect in an autocrining manner.
(29) Preparation of recombinant amino- and carboxyl-terminal RNF43 protein
To generate a specific antibody against RNF43, a plasmid expressing Nus-tagged
RNF43 protein was constructed (Figure 30A). Upon transformation of the plasmid
into E.
coli BL21trxB(DE3)pLysS cell, production of a recombinant protein in the
bacterial extract
with the expected size was observed by SDS-PAGE (Figure 30B and 30C).
(30) Identification of RNF43-interacting proteins by yeast two-hybrid
screening system
To clarify the oncogenic mechanism of RNF43, RNF43-interacting proteins were
searched using yeast two-hybrid screening system. Among the identified
positive clones,
NOTCH2 or STRIN interacted with RNF43 by simultaneous transformation of an
yeast
cell with pAS2.1-RNF43 and pACT2-NOTCH2 (Figure 31B), or pAS2.1-RNF43 and
pACT2-STRIN (Figure 32B). The regions responsible for the interaction in
NOTCH2
and STRIN are indicated in Figure 31A and Figure 32A, respectively.
(31) Prediction of HLA-A24 binding peptides derived from RNF43
The amino acid sequence of RNF43 was scanned for peptides with a length of 9
or
10 amino acids which peptides bind to HLA-A24 using the binding prediction
soft
Table 3 shows the predicted peptides (SEQ ID NOs:71-90) in order of high
binding
affinity. Twenty peptides in total were selected and examined as described
below.


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Table 3

Predicted RNF43peptides binding to HL.A-A24

Start AA mgmnm Start AA sequexne
p tnn (9mers) Ending affinity *' position (10noexa) Bindingaffinity
RNF43-331 SYQEPGRR.L 360 RNF43-449 SYC!'IILSGYL 200
RNF43-350 HYHLPAAYL 200 RNF43-350 HYHLPAAYLL 200
RNF43-639 LFNLQ:KSSL 30 RNF43-718 CYSNSQPVWL 200
RNF43-24 GFGRTGLVL 20 RNF43-209 IFV1U SVL 36
RNF43-247 RYQASCRQA 15 RNF43-313 VFNITEGDSF 15
RNF43-397 RAPGEQQRL 14 RNF43-496 TFCSSLSSDF 12
RNF43-114 RAPRPCISL 12 RNF43-81 KLMQSHP'LYL 12
RNF43.368 RPPRPGPFL 12 RNF43-54 KMDPTGKLNL 9
RNP43-45 KAVIRVIPL 12 RNF43-683 HY1PSVAYPW 8
RNP43-721 NSQPVWLCL 10 RNF43-282 GQELLRVISCL 4

In the table, start position indicates the location of amino acids from the N-
terminus of
RNF43.
(32) Stimulation of T cells using the predicted peptides
CTLs against these peptides derived from RNF43 were generated according to the
method described in the "Materials and Methods". Resulting CTLs showing
detectable
cytotoxic activity were expanded, and CTL lines were established.
The cytotoxic activities of CTL lines induced by 9 mer-peptide (SEQ ID NOs:
71-80) stimulation are shown in Table 4.


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Table 4 Cytotoxicity of CTL lines (9mer)

Start AA Binding Cytotoxicity
position sequences affinity X 20 X 2 Established
Pep(+) Pep(-) Pep(+) Pep(-) CTL clones
RNF43-331 SYQEPGRRL 360.0 2% 1% 0% 0%
RNF43-350 HYHLPAAYL 200.0 26% 17% 5% 4%
RNF43-639 WNLQKSSL 30.0 42% 33% 7% 5% 1
RNF43-24 GFGRTGLVL 20.0 8% 9% 1% 2%
RNF43-247 RYQASCRQA 15.0 71% 82% 28% 16%
RNF43-397 RAPGEQQRL 14.4 41% 32% 15% 15%
RNF43-114 RAPRPCLSL 12.0 23% 26% 6% 9%
RNF43-368 RPPRPGPFL 12.0 1% 0% 0% 0%
RNF43-45 KAVIRVIPL 12.0 NE
RNF43-721 NSQPVWLCL 10.0 68% 0% 26% 0% 13

No establishment of CTL lines

CTL lines stimulated with RNF43-350 (HYHLPAAYL) (SEQ ID NO: 72), RNF43-639
(LFNLQKSSL) (SEQ ID NO: 73), and RNF43-721 (NSQPVWLCL) (SEQ ID NO: 80)
showed higher cytotoxic activities on the target that were pulsed with
peptides compared
with those on target that was not pulsed with any of the peptides. Starting
from these
CTLs, one CTL clone was established with RNF43-639 and 13 CTL clones were
established with RNF43-721.
The CTL line stimulated with RNF43-721 showed a potent cytotoxic activity on
the peptide-pulsed target without showing any significant cytotoxic activity
on target that
was not pulsed with any of the peptides (Figure 33).
The results obtained by examining the cytotoxic activity of CTL lines
stimulated
with the 10 mer-peptides (SEQ ID NOs: 81-90) are shown in Table 5.


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Table 5 Cytotoxicity of CTL lines (10mer)

Start AA Binding Cytotoxicity
position sequences affinity X 20 X2 Established
Pep(+) Pep(-) Pep(+) Pep(-) CTL clones
RNF43-449 SYCTERSGYL 200.0 1% 1% 0% 0%
RNF43-350 HYBLPAAYLL 200.0 NE
RNF43-718 CYSNSQPVWL 200.0 NE
RNF43-209 IFVLILASVL 36.0 Not synthesis
RNF43-313 VFNLTEGDSF 15.0 Not synthesis
RNF43-496 TFCSSLSSDF 12.0 8% 9% 0% 0%
RNF43-81 KLMQSHPLYL 12.0 10% 5% 2% -3%
RNF43-54 KMDPTGKLNL 9.6 5% 2% 0% -1%
RNF43-683 HYTPSVAYPW 8.4 0% 0% 0% -1%
RNF43-282 GQELRVISCL 8.4 NE

NE:No establishment of C-M lines

CTL lines stimulated with RNF43-81 (KLMQSHPLYL) (SEQ ID NO: 87) or RNF43-54
(KMDPTGKLNL) (SEQ ID NO: 88) showed modest cytotoxic activity on the
5 peptide-pulsed target compared with that on the target that was not pulsed
with any of the
peptides.
(33) Establishment of CTL clones
CTL clones were propagated from the CTL lines described above using the
limiting dilution method. 13 CTL clones against RNF43-721 and 1 CTL clone
against
10 RNF43-639 were established (see Table 4 supra). The cytotoxic activity of
RNF43-721
CTL clones is shown in Figure 34. Each CTL clone had a very potent cytotoxic
activity
on the peptide-pulsed target without showing any cytotoxic activity on the
target that was
not pulsed with any of the peptides.
(34) Cyttotoxic activity against colorectal cancer cell lines endogenously
expressing RNF43
15 as target
The CTL clones raised against predicted peptides were examined for their
ability
to recognize and kill tumor cells that endogenously express RNF43. Figure35
shows the
results of CTL Clone 45 raised against RNF43-721. CTL Clone 45 showed a potent
cytotoxic activity on HT29 and WiDR both expressing RNF43 and HLA-A24. On the
20 other hand, CTL Clone 45 did not show any cytotoxic activity on either
HCT116
(expressing RNF43 but not HLA-A24) or TISI (expressing HLA-A24 but not RNF43).
Moreover, CTL Clone 45 did not show any cytotoxic activity on irrelevant
peptide pulsed


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TISI and SNU-C4 that express RNF43 but little HLA-A24 (data not shown).
(35) Characterization of established CTLs
A cold target inhibition assay was performed to confirm the specificity of
RNF43-721 CTL Clone. HT29 cells labeled by 51Cr were used as a hot target,
while TISI
cells pulsed with RNF43-721 without 51Cr labeling were used as a cold target.
When
TISI pulsed with RNF43-721 was added in the assay at various ratios, specific
cell lysis
against the HT29 cell target was significantly inhibited, (Figure 36). This
result is
indicated as a percentage of specific lysis at the E/T ratio of 20. To examine
the
characteristics of the CTL clone raised against RNF43 peptide, antibodies
against
HLA-Class I, HLA-Class II, CD3, CD4 and CD8 were tested for their ability to
inhibit the
cytotoxic activity of the CTL clone. The cytotoxicity of the CTL clone on the
WiDR cell
target was significantly inhibited when anti-HLA-Class I, CD3 and CD8
antibodies were
used (Figure 37). The result indicates that the CTL clone recognizes the RNF43
derived
peptide via HLA-Class I, CD3 and CD8.
(36) Homology analysis of RNF43-721 peptide
The CTL clones established against RNF43-721 showed a very potent cytotoxic
activity. This result may indicate that the sequence of RNF43-721 is
homologous to the
peptides derived from other molecules which are known to sensitize human
immune
system. To exclude this possibility, homology analysis of RNF43-721 was
performed
using BLAST. No sequence completely matching or highly homologous to RNF43-721
was found among the molecules listed in BLAST (Table 6).

Table 6
Homology analysis of RNF43-721
Identification(9/9) 0
Identification(819) 0
Identification(7/9) 0
Identification(619) 2

These results indicate that the sequence of RNF43-721 is unique and there is
little
possibility for the CTL clones established with RNF43-721 to raise immunologic
response
to other molecules.


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(37) Modification of RNF43-721 to improve the efficacy of epitope presentation
To improve the efficacy of peptide presentation, RNF43-721 peptide were
modified at amino acid alternations on the anchor site. The modification was
expected to
improve the binding affinity of the peptide to the HLA Class I molecule. Table
7
demonstrates a better binding affinity to HLA-A24 molecule of RNF43-721 with
alternations of amino acids at position 2 (SEQ ID NOs: 91 and 92).

Table 7

Predicted binding capacities of the peptides modified
from the RNF43-721 native peptide
Sequence Score Rank*
NSQPVWLCL 10.08 10
NFQPVWLCL 50.40 3
NYQPVWLCL 504.00 1
* In HLA-A24 restricted 9mer peptides

(38) Prediction of candidate peptides derived from RNF43
Table 8 shows candidate peptides (SEQ ID NOs: 87, and 93-111) in order of high
binding affinity.


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Table 8

RNF43:Prediction of epitope peptides (HLA-A*0201)
9mer 10mer
No Starting Sequences Score No Starting Sequences Score
position position
1 60 KLNLTLEGV 274.3 11 81 KLMQSHPLYL 1521.5
2 8 QLAALWPWL 199.7 12 357 YLLGPSRSAV 1183.7
3 82 LMQSHPLYL 144.2 13 202 LMTVVGTIFV 469.6
4 358 LLGPSRSAV 118.2 14 290 CLHEFHRNCV 285.1
11 A.LWPWLLMA 94.8 15 500 SLSSDFDPLV 264.2
6 15 WLLMATLQA 84.5 16 8 QLAAIWPWLL 160.2
7 200 WILMTVVGT 40.1 17 11 ALWPWLLMAT 142.2
18 7 LQLAALWPWL 127.3
8 171 KLMEFVYKN 34.5
19 726 WLCLTPRQPL 98.2
9 62 NLTLEGVFA 27.3 20 302 WLHQHRTCPL 98.2
156 GLTWPVVLI 23.9

Twenty peptides in total were selected and examined as described below.
(39) Stimulation of T cells using candidate peptides
5 Lymphoid cells were cultured using the candidate peptides derived from RNF43
according to the method described in the "Materials and Methods". Resulting
lymphoid
cells showing detectable cytotoxic activity were expanded, and CTL lines were
established.
The cytotoxic activities of CTL lines induced by the stimulation using 9 mer-
peptides
(SEQ ID NOs: 93-102) are shown in Table 9.


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Table 9

Cytotoxicity of CTL lines (HLA-A*0201 9mer)

Start AA Binding Cytotoxicity(%)
position sequences affinity X 20 X2
Pep(+) Pep(-) Pep(+) Pep(-)
RNF43-60 KLNLTLEGV 274.3 -2.1 0.2 -1.6 0.0
RNF43- 8 QLAALWPWL 199.7 3.5 0.0 0.0 1.0
RNF43-82 LMQSHPLYL 144.2 1.7 1.2 0.0 -0.4
RNF43-358 LLGPSRSAV 118.2 -0.4 -0.7 0.0 -0.8
RNF43-11 ALWPWLLMA 94.8 90.2 1.5 45.4 1.3
RNF43-15 WLLMATLQA 84.5 -0.2 0.0 -0.4 -0.9
RNF43-200 WILMTVVGT 40.1 Not Synthesis
RNF43-171 KLMEFVYKN 34.5 2.6 0.0 1.1 -0.5
RNF43-62 NLTLEGVFA 27.3 Not Synthesis
RNF43-156 GLTWPVVLI 23.9 -0.4 0.7 -0.5 -0.3

NE:No establishment of CTL lines

CTL lines induced with RNF43-11-9 (ALWPWLLMA) (SEQ ID NO: 97) showed higher
cytotoxic activities on the target pulsed with peptides compared with those on
the target
that was not pulsed with any of the peptides. Starting from these CTLs, four
CTL clone
were established with RNF43-11-9. The CTL line stimulated with RNF43-11-9
showed a
potent cytotoxic activity on the peptide-pulsed target without showing any
significant
cytotoxic activity on the target that was not pulsed with any of the peptides
(Figure 38A).
The results of examination on the cytotoxic activity of CTL lines induced with
the
10 mer-peptides (SEQ ID NOs: 87, and 103-111) are shown in Table 10.


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Table 10

Cytotoxicity of CTL lines (HLA-A*0201 l0mer)

Start AA Binding Cytotoxicity(%)
position sequences affinity X 20 X 2
Pep(+) Pep(-) Pep(+) Pep(-)
RNF43-81 KLMQSHPLYL 1521.5 18.0 27.6 6.3 8.3
RNF43-357 YLLGPSRSAV 1183.7 18.2 15.4 3.7 3.0
RNF43-202 LMTVVGTIFV 469.6 Not Synthesis
RNF43-290 CLHEFHRNCV 285.1 9.6 9.7 2.7 3.7
RNF43-500 SLSSDFDPLV 264.2 NE
RNF43-8 QLAAIWPWLL 160.2 6.7 9.0 1.1 1.3
RNF43-11 ALWPWLLMAT 142.2 91.5 27.1 40.5 4.3
RNF43-7 LQLAALWPWL 127.3 NE
RNF43-726 WLCLTPRQPL 98.2 NE
RNF43-302 WLHQHRTCPL 98.2 7.4 6.1 1.5 2.2

NE:No establishment of CTL lines

CTL lines induced with RNF43-11-10 (ALWPWLLMAT) (SEQ ID NO: 108) showed a
higher cytotoxic activity on the peptide-pulsed target compared with that on
the target that
5 was not pulsed with any of the peptides (Figure 38B).
(40) Establishment of CTL clones
CTL clones were propagated from the CTL lines described above using the
limiting dilution method. Four CTL clones against RNF43-11-9 were established
(see
Table 9 supra). The cytotoxic activity of RNF43 peptides-derived CTL clones is
shown
10 in Figure 39A and 39B. Each CTL clone had a very potent cytotoxic activity
on the
peptide-pulsed target without showing any cytotoxic activity on the target
that was not
pulsed with any of the peptides.
(41) Cytotoxic activity against colorectal cancer cell lines endogenously
expressing RNF43
as targets
15 The CTL clones raised against the predicted peptides were examined for
their
ability to recognize and kill tumor cells that endogenously express RNF43.
Figure 40A
and 40B show the results obtained for the CTL clones raised against RNF43
derived
peptides. The CTL Clones showed a potent cytotoxic activity on DLD-1 which
expresses
RNF43 and HLA-A*0201, but none on HT29 which expresses RNF43 but not


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81
HLA-A*0201.
(42) Specificity of CTL clones
A cold target inhibition assay was performed to confirm the specificity of
RNF43-5-11(9mer) CTL Clone. HCT116 cells labeled with 51Cr were used as a hot
target,
while T2 cells pulsed with RNF43-5 without 51Cr labeling were used as a cold
target.
Specific cell lysis of the HCT-116 cell target was significantly inhibited,
when T2 pulsed
with RNF43-5 was added in the assay at various ratios (Figure 41).

Industrial Applicability
The expression of novel human genes CXADRLI and GCUD1 is markedly
elevated in gastric cancer as compared to non-cancerous stomach tissues. On
the other
hand, the expression of novel human gene RNF43 is markedly elevated in
colorectal
cancers as compared to non-cancerous mucosal tissues. Accordingly, these genes
may
serve as a diagnostic marker of cancer and the proteins encoded thereby may be
used in
diagnostic assays of cancer.
The present inventors have also shown that the expression of novel protein
CXADRL1, GCUD1, or RNF43 promotes cell growth whereas cell growth is
suppressed
by antisense oligonucleotides or small interfering RNAs corresponding to the
CXADRLI,
GCUD1, or RNF43 gene. These findings suggest that each of CXADRL1, GCUD1, and
RNF43 proteins stimulate oncogenic activity. Thus, each of these novel
oncoproteins is
useful targets for the development of anti-cancer pharmaceuticals. For
example, agents
that block the expression of CXADRL1, GCUD1, or RNF43, or prevent its activity
may
find therapeutic utility as anti-cancer agents, particularly anti-cancer
agents for the
treatment of gastric and colorectal cancers. Examples of such agents include
antisense
oligonucleotides, small interfering RNAs, and antibodies that recognize
CXADRL1,
GCUD1, or RNF43.
Furthermore, the present inventors have shown that CXADRL1 interacts with
AIP1. It is expected that the cell proliferating activity of CXADRL1 is
regulated by its
binding to AIP1. Thus, agents that inhibit the activity of the formation of a
complex
composed of CXADRLI and AIP1 may also find utility in the treatment and
prevention of
cancer, specifically colorectal, lung, gastric, and liver cancers.
Alternatively, the present
inventors have shown that RNF43 interacts with NOTCH2 or STRIN. It is expected
that
the cell proliferating activity of RNF43 is regulated by its binding to NOTCH2
or STRIN.
Thus, agents that inhibit the activity of the formation of a complex composed
of RNF43
and NOTCH2 or STRIN may also find utility in the treatment and prevention of
cancer,
specifically colorectal, lung, gastric, and liver cancers.


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While the invention has been described in detail and with reference to
specific
embodiments thereof, it will be apparent to one skilled in the art that
various changes and
modifications can be made therein without departing from the spirit and scope
of the
invention.


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83
SEQUENCE LISTING
<110> ONCOTHERAPY SCIENCE, INC.

<120> GENES AND POLYPEPTIDES RELATING TO HUMAN COLON CANCERS
<130> 12871-108

<140> CA 2,488,404
<141> 2003-06-03
<150> US 60/386,985
<151> 2002-06-06
<150> US 60/415,209
<151> 2002-09-30
<150> US 60/451,013
<151> 2003-02-28
<160> 194

<170> Patentln version 3.1
<210> 1
<211> 3423
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (238)..(1533)
<223>

<400> 1
gctggcgagc ccggaacgcc tctggtcaca gctcagcgtc cgcggagccg ggcggcgctg 60
gagctgcact tggctcgtct gtgggtctga cagtcccagc tctgcgcggg gaacagcggc 120
ccggagctgg gtgtgggagg accaggctgc cccaagagcg cggagactca cgcccgctcc 180
tctcctgttg cgaccgggag ccgggtagga ggcaggcgcg ctccctgcgg ccccggg 237
atg act tct cag cgt tcc cct ctg gcg cct ttg ctg ctc ctc tct ctg 285
Met Thr Ser Gln Arg Ser Pro Leu Ala Pro Leu Leu Leu Leu Ser Leu
1 5 10 15
cac ggt gtt gca gca tcc ctg gaa gtg tca gag agc cct ggg agt atc 333
His Gly Val Ala Ala Ser Leu Glu Val Ser Glu Ser Pro Gly Ser Ile
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cag gtg gcc cgg ggt cag cca gca gtc ctg ccc tgc act ttc act acc 381
Gln Val Ala Arg Gly Gln Pro Ala Val Leu Pro Cys Thr Phe Thr Thr
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agc get gcc ctc att aac ctc aat gtc att tgg atg gtc act cct ctc 429
Ser Ala Ala Leu Ile Asn Leu Asn Val Ile Trp Met Val Thr Pro Leu
50 55 60


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84
tcc aat gcc aac caa cct gaa cag gtc atc ctg tat cag ggt gga cag 477
Ser Asn Ala Asn Gln Pro Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln
65 70 75 80
atg ttt gat ggt gcc ccc cgg ttc cac ggt agg gta gga ttt aca ggc 525
Met Phe Asp Gly Ala Pro Arg Phe His Gly Arg Val Gly Phe Thr Gly
85 90 95
acc atg cca get acc aat gtc tct atc ttc att aat aac act cag tta 573
Thr Met Pro Ala Thr Asn Val Ser Ile Phe Ile Asn Asn Thr Gln Leu
100 105 110
tca gac act ggc acc tac cag tgc ctg gtc aac aac ctt cca gac ata 621
Ser Asp Thr Gly Thr Tyr Gln Cys Leu Val Asn Asn Leu Pro Asp Ile
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ggg ggc agg aac att ggg gtc acc ggt ctc aca gtg tta gtt ccc cct 669
Gly Gly Arg Asn Ile Gly Val Thr Gly Leu Thr Val Leu Val Pro Pro
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tct gcc cca cac tgc caa atc caa gga tcc cag gat att ggc agc gat 717
Ser Ala Pro His Cys Gln Ile Gln Gly Ser Gln Asp Ile Gly Ser Asp
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gtc atc cta ctc tgt agc tca gag gaa ggc att cct cga cca act tac 765
Val Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr
165 170 175
ctt tgg gag aag tta gac aat acc ctc aaa cta cct cca aca get act 813
Leu Trp Glu Lys Leu Asp Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr
180 185 190
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Gln Asp Gln Val Gln Gly Thr Val Thr Ile Arg Asn Ile Ser Ala Leu
195 200 205

tct tca ggt ttg tac cag tgc gtg get tct aat get att gga acc agc 909
Ser Ser Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala Ile Gly Thr Ser
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acc tgt ctt ctg gat ctc cag gtt att tca ccc cag ccc agg aac att 957
Thr Cys Leu Leu Asp Leu Gln Val Ile Ser Pro Gln Pro Arg Asn Ile
225 230 235 240
gga cta ata get gga gcc att ggc act ggt gca gtt att atc att ttt 1005
Gly Leu Ile Ala Gly Ala Ile Gly Thr Gly Ala Val Ile Ile Ile Phe
245 250 255
tgc att gca cta att tta ggg gca ttc ttt tac tgg aga agc aaa aat 1053
Cys Ile Ala Leu Ile Leu Gly Ala Phe Phe Tyr Trp Arg Ser Lys Asn
260 265 270
aaa gag gag gaa gaa gaa gaa att cct aat gaa ata aga gag gat gat 1101
Lys Glu Glu Glu Glu Glu Glu Ile Pro Asn Glu Ile Arg Glu Asp Asp
275 280 285

ctt cca ccc aag tgt tct tct gcc aaa gca ttt cac act gag att tcc 1149
Leu Pro Pro Lys Cys Ser Ser Ala Lys Ala Phe His Thr Glu Ile Ser
290 295 300


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tcc tcg gac aac aac aca cta acc tct tcc aat gcc tac aac agt cga 1197
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Tyr Trp Ser Asn Asn Pro Lys Val His Arg Asn Thr Asp Ser Val Ser
325 330 335
cac ttc agt gac ttg ggc caa tct ttc tct ttc cac tca ggc aat gcc 1293
His Phe Ser Asp Leu Gly Gln Ser Phe Ser Phe His Ser Gly Asn Ala
340 345 350
aac ata cca tcc att tat get aat ggg acc cat ctg gtc ccg ggt caa 1341
Asn Ile Pro Ser Ile Tyr Ala Asn Gly Thr His Leu Val Pro Gly Gln
355 360 365

cat aag act ctg gta gtg aca gcc aac aga ggg tca tca cca cag gtg 1389
His Lys Thr Leu Val Val Thr Ala Asn Arg Gly Ser Ser Pro Gln Val
370 375 380

atg tcc agg agc aat ggc tca gtc agt agg gag cct cgg cct cca cac 1437
Met Ser Arg Ser Asn Gly Ser Val Ser Arg Glu Pro Arg Pro Pro His
385 390 395 400
act cat tcc tac acc atc agc cac gca aca ctg gaa cga att ggt gca 1485
Thr His Ser Tyr Thr Ile Ser His Ala Thr Leu Glu Arg Ile Gly Ala
405 410 415
gta cct gtc atg gta cca gcc cag agt cgg gcc ggg tcc ttg gta tag 1533
Val Pro Val Met Val Pro Ala Gln Ser Arg Ala Gly Ser Leu Val
420 425 430
gacatgagga aatgttgtgt tcagaaatga ataaatggaa tgccctcata caagggggag 1593
ggtggggtgg ggagtgctgg gaaagaaaca cttccttata attatattag taaaatgcac 1653
aaagaagaag gcagtgctgt tacttggcca ctaagatgtg taaaatggac tgaaatgctc 1713
catcatgaag acttgcttcc ccaccaaaga tgtcctggga ttctgctgga tctcaaagat 1773
gtgccaagcc aaggaaaaag atacaagagc agaatagtac ttaaaatcca aactgccgcc 1833
cagatgggct tgttcttcat gcctaactta ataattttta agagattaaa gtgccagatg 1893
gagtttaaat attgaaatta ttttaaaagg taggtgtctt taagaaaata acaagcaacc 1953
ctgtgatatg ttccgtctct cccaattccc tcgttatata gagggcttaa tggtataaat 2013
ggttaatatt ggtcccaaca gggctgactc ttctatcata taatcaaaac tttttacatg 2073
agcaaaattc agtaagaaat gggggaagac aaaggaaacg tctttgagaa gccccttcat 2133
atttatttat ttatctcttc ctgaaccatg aatttcatat gtggaatatt gctatattga 2193
cagattcttg cctgtctgtg ttattctagg atctgttaca ggtccatggc aattactgtt 2253
tattttttcc tggaaaaata tttttttata aaaggctttt tttttttttt aaatacatga 2313


CA 02488404 2007-06-05

86
gaggcattgg gctaagaaag aaaagactgt tgtataatac cttgttcaat ggttgtattt 2373
agtgagctca tagaggtcca tcatatcatg accgagctag gttgtgtggg caggaaggta 2433
gggctaaggg gttgtagcct tgctgggcag cctctcagag caaggttgtt cagatctccc 2493
ttgctattac agtaggttac tattaatgag ggcagcacct gatgcctttt gtactgaggt 2553
atgtaacttt ctccttattt gacaagtaga agttaactta cttgtcaggg agggcagacg 2613
tttttttgtt ctgtttcgtt tttcaaaata atgctttttg caaaagaggt aagactgaga 2673
ctaaaggtgt tatcttctgg tgtgctcctg gaagtgtcta ccctacattt gtgtcagctc 2733
agggttgcag tgttgcccag atgcatttta catcactgta aagagattac ttttgtggtt 2793
actacctggc ttggctggcc ttgcggttca ccagattaat ttacaaactc ccccacttta 2853
ttttgtgcta tgtagatctg gccatacttg cattagtgac tgtcttgcct taaccacact 2913
taagcaaccc acaaatttct tctcagattt gtttcctaga ttacttatga tactcatccc 2973
atgtctcaat aagagtgtct tttctttctg gatgtgttct cttactccct cttaccacca 3033
tactttttgc tctcttctcc tgcaagcgta gtcttcacag ggagtggctt cctgacattt 3093
ttttcagtta tgtgaatgaa tggaaaccaa cagctgctgc aaacactgtt tttccaagaa 3153
ggctacactc agaacctaac cattgccaac catttcagta ttgataaaaa gctgaattta 3213
ctttagcatt acttattttt ttttccattt gatggttctt actttgtaaa aatttaaata 3273
aatgaatgtc tatacttttt ataaagaaaa gtgaaaatac catgacactg aaaagatgat 3333
gctatcagat gctgtttaga aagcatttat cttgcatttc tttattcttt ctaattatct 3393
aaaattcaat aaaattttat tcatataaaa 3423
<210> 2
<211> 431
<212> PRT
<213> Homo sapiens
<400> 2
Met Thr Ser Gln Arg Ser Pro Leu Ala Pro Leu Leu Leu Leu Ser Leu
1 5 10 ].5
His Gly Val Ala Ala Ser Leu Glu Val Ser Glu Ser Pro Gly Ser Ile
20 25 30
Gln Val Ala Arg Gly Gln Pro Ala Val Leu Pro Cys Thr Phe Thr Thr
35 40 45

Ser Ala Ala Leu Ile Asn Leu Asn Val Ile Trp Met Val Thr Pro Leu
50 55 60
Ser Asn Ala Asn Gln Pro Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln
65 70 75 80


CA 02488404 2007-06-05

87
Met Phe Asp Gly Ala Pro Arg Phe His Gly Arg Val Gly Phe Thr Gly
85 90 95

Thr Met Pro Ala Thr Asn Val Ser Ile Phe Ile Asn Asn Thr Gln Leu
100 105 110
Ser Asp Thr Gly Thr Tyr Gln Cys Leu Val Asn Asn Leu Pro Asp Ile
115 120 125
Gly Gly Arg Asn Ile Gly Val Thr Gly Leu Thr Val Leu Val Pro Pro
130 135 140

Ser Ala Pro His Cys Gln Ile Gln Gly Ser Gln Asp Ile Gly Ser Asp
145 150 155 160
Val Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr
165 170 175

Leu Trp Glu Lys Leu Asp Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr
180 185 190
Gln Asp Gln Val Gln Gly Thr Val Thr Ile Arg Asn Ile Ser Ala Leu
195 200 205
Ser Ser Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala Ile Gly Thr Ser
210 215 220

Thr Cys Leu Leu Asp Leu Gln Val Ile Ser Pro Gln Pro Arg Asn Ile
225 230 235 240
Gly Leu Ile Ala Gly Ala Ile Gly Thr Gly Ala Val Ile Ile Ile Phe
245 250 255

Cys Ile Ala Leu Ile Leu Gly Ala Phe Phe Tyr Trp Arg Ser Lys Asn
260 265 270
Lys Glu Glu Glu Glu Glu Glu Ile Pro Asn Glu Ile Arg Glu Asp Asp
275 280 285
Leu Pro Pro Lys Cys Ser Ser Ala Lys Ala Phe His Thr Glu Ile Ser
290 295 300

Ser Ser Asp Asn Asn Thr Leu Thr Ser Ser Asn Ala Tyr Asn Ser Arg
305 310 315 320
Tyr Trp Ser Asn Asn Pro Lys Val His Arg Asn Thr Asp Ser Val Ser
325 330 335

His Phe Ser Asp Leu Gly Gln Ser Phe Ser Phe His Ser Gly Asn Ala
340 345 350
Asn Ile Pro Ser Ile Tyr Ala Asn Gly Thr His Leu Val Pro Gly Gln
355 360 365
His Lys Thr Leu Val Val Thr Ala Asn Arg Gly Ser Ser Pro Gln Val
370 375 380


CA 02488404 2007-06-05

88
Met Ser Arg Ser Asn Gly Ser Val Ser Arg Glu Pro Arg Pro Pro His
385 390 395 400
Thr His Ser Tyr Thr Ile Ser His Ala Thr Leu Glu Arg Ile Gly Ala
405 410 415

Val Pro Val Met Val Pro Ala Gln Ser Arg Ala Gly Ser Leu Val
420 425 430
<210> 3
<211> 4987
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (60)..(1304)
<223>

<400> 3
gcggccgcag cctcagcacc gcagagcgga gagcggagcc cggagcccgc cgccccagg 59
atg get gca get cct cca agt tac tgt ttt gtt gcc ttc cct cca cgt 107
Met Ala Ala Ala Pro Pro Ser Tyr Cys Phe Val Ala Phe Pro Pro Arg
1 5 10 1.5
get aag gat ggt ctg gtg gta ttt ggg aaa aat tca gcc cgg ccc aga 155
Ala Lys Asp Gly Leu Val Val Phe Gly Lys Asn Ser Ala Arg Pro Arg
20 25 30
gat gaa gtg caa gag gtt gtg tat ttc tcg get get gat cac gaa ccg 203
Asp Glu Val Gln Glu Val Val Tyr Phe Ser Ala Ala Asp His Glu Pro
35 40 45

gag agc aag gtt gag tgc act tac att tca atc gac caa gtt cca agg 251
Glu Ser Lys Val Glu Cys Thr Tyr Ile Ser Ile Asp Gln Val Pro Arg
50 55 60

acc tat gcc ata atg ata agc aga ccc gcc tgg ctc tgg gga gca gaa 299
Thr Tyr Ala Ile Met Ile Ser Arg Pro Ala Trp Leu Trp Gly Ala Glu
65 70 75 80
atg gga gcc aat gaa cat gga gtg tgc ata gcc aat gaa gcc atc aac 347
Met Gly Ala Asn Glu His Gly Val Cys Ile Ala Asn Glu Ala Ile Asn
85 90 95
acc aga gag cca get gcc gag ata gaa gcc ttg ctg ggg atg gat ctg 395
Thr Arg Glu Pro Ala Ala Glu Ile Glu Ala Leu Leu Gly Met Asp Leu
100 105 110
gtc agg ctt ggt tta gaa aga ggg gaa aca get aaa gaa gcc tta gat 443
Val Arg Leu Gly Leu Glu Arg Gly Glu Thr Ala Lys Glu Ala Leu Asp
115 120 125

gtc att gtc tcc ttg ttg gaa gaa cat gga caa ggt ggg aat tac ttt 491
Val Ile Val Ser Leu Leu Glu Glu His Gly Gln Gly Gly Asn Tyr Phe
130 135 140


CA 02488404 2007-06-05

89
gaa gat gca aac tcc tgc cac agc ttc caa agt gca tat ctg att gtg 539
Glu Asp Ala Asn Ser Cys His Ser Phe Gln Ser Ala Tyr Leu Ile Val
145 150 155 160
gat cgt gat gaa gcc tgg gtg ctc gag acc ata ggg aag tac tgg get 587
Asp Arg Asp Glu Ala Trp Val Leu Glu Thr Ile Gly Lys Tyr Trp Ala
165 170 175
gcc gag aaa gtc aca gag gga gtg agg tgc att tgc agt cag ctt tcg 635
Ala Glu Lys Val Thr Glu Gly Val Arg Cys Ile Cys Ser Gln Leu Ser
180 185 190
ctc acc act aag atg gat gca gag cat ccg gaa ctc agg agt tac get 683
Leu Thr Thr Lys Met Asp Ala Glu His Pro Glu Leu Arg Ser Tyr Ala
195 200 205

cag agc caa ggt tgg tgg acg gga gag ggc gag ttc aat ttt tcc gaa 731
Gln Ser Gln Gly Trp Trp Thr Gly Glu Gly Glu Phe Asn Phe Ser Glu
210 215 220

gtc ttt tct cca gtt gag gat cat cta gac tgc ggt get ggc aaa gac 779
Val Phe Ser Pro Val Glu Asp His Leu Asp Cys Gly Ala Gly Lys Asp
225 230 235 240
agc tta gaa aaa caa gaa gaa agc atc aca gtg cag act atg atg aac 827
Ser Leu Glu Lys Gln Glu Glu Ser Ile Thr Val Gln Thr Met Met Asn
245 250 255
acc tta cgg gac aaa gcc agc gga gtg tgc ata gac tct gag ttt ttc 875
Thr Leu Arg Asp Lys Ala Ser Gly Val Cys Ile Asp Ser Glu Phe Phe
260 265 270
ctc acc aca gcc agt gga gtg tct gtc ctg ccg cag aat aga agc tct 923
Leu Thr Thr Ala Ser Gly Val Ser Val Leu Pro Gln Asn Arg Ser Ser
275 280 285

ccg tgc att cac tac ttc act gga acc cct gat cct tcc agg tcc ata 971
Pro Cys Ile His Tyr Phe Thr Gly Thr Pro Asp Pro Ser Arg Ser Ile
290 295 300

ttc aag cct ttc atc ttt gtt gat gac gta aaa ctt gtc ccc aaa aca 1019
Phe Lys Pro Phe Ile Phe Val Asp Asp Val Lys Leu Val Pro Lys Thr
305 310 315 320
cag tct ccc tgt ttt ggg gat gac gac cct gcc aaa aag gag cct cgg 1067
Gln Ser Pro Cys Phe Gly Asp Asp Asp Pro Ala Lys Lys Glu Pro Arg
325 330 335
ttc cag gag aaa cca gac cgc cgg cat gag ctg tac aaa gcc cac gag 1115
Phe Gln Glu Lys Pro Asp Arg Arg His Glu Leu Tyr Lys Ala His Glu
340 345 350
tgg gca cgt gcc atc atc gaa agt gac cag gag caa ggt cgc aag ctg 1163
Trp Ala Arg Ala Ile Ile Glu Ser Asp Gln Glu Gln Gly Arg Lys Leu
355 360 365

agg agc acc atg ctg gag ctg gag aag caa ggc ctg gaa gcc atg gaa 1211
Arg Ser Thr Met Leu Glu Leu Glu Lys Gln Gly Leu Glu Ala Met Glu
370 375 380


CA 02488404 2007-06-05

gaa atc ctg acc agc tcc gag cca ctg gac cct gcg gaa gtg ggg gac 1259
Glu Ile Leu Thr Ser Ser Glu Pro Leu Asp Pro Ala Glu Val Gly Asp
385 390 395 400
ctt ttc tat gac tgt gtt gac acg gag att aag ttc ttt aag tga 1304
Leu Phe Tyr Asp Cys Val Asp Thr Glu Ile Lys Phe Phe Lys
405 410

agtaagcgtt ccctttcccc ttcttattta agacttccca ccttactaaa ttaccagcaa 1364
aacaaaccac tctcctgttt gagtaaaatg agaaagttaa tatgtggcct ccttttctga 1424
agccagatca aactgttacc ttgtgttcca ccttgaatct cacagcgtcc ccttctgcaa 1484
tgtaggtctc cttcctgtgc agtgtaacat gtatcccgtt gcctgttgtt cggttgtgtg 1544
actaattgtg gattttaagc tgctattatt gtatttcagt ggcaatggac acattagcct 1604
tttacaagag gactagagtt catcaagcct tgaaaggcag gcttcacagt gccgagttgg 1664
cgggaaaagc aaattctttt gaagtcttag tctttccctc agtagcggtt tctttcaggt 1724
taacaagagg catttgtgca cacacacagg gctcttgtgt gtgttgtcaa ggggaccctc 1784
cgtggcctcc cgtgagtgca tgcctgtagt gcacagtgtc tctacaggtg tcttctgggg 1844
ggcagaacca attggaagga agaaagggac ccctctccag tcctggctcc ttcctacatc 1904
ctgggctcct gaagaagctg tcttcccatt ttccatgcgc tgtgcttatg tgtggtggac 1964
tgcagagctg cttccactta caggagagct gataatttgt tagctggaac ctattcactt 2024
ccgagattca gacatagcca tgctggtggc cttctgaatc actgcatgga tgtcccagga 2084
ggcagctctc cccacacagc agcacagcca tcacaggatt ccttgtgtag aaatgattcc 2144
cagtctagtt accaacagct agtctaggag taattgaatg gccctatggc acagttccac 2204
ccacagagta gtgaatctct cagccaagga gggaaagaaa aggaagaact cttgactatt 2264
tagattctag ttaaatatct ggaatcctag cagtcactac attatctcag cagagagact 2324
ttaattaaac tgatttgttt ccaatgtcgg gttcacttaa aggatttgac ttaccaccag 2384
agcatagaaa agcatgcaag gaagaccaga tgggcttagc attgggaaga cagagggcaa 2444
ggaggtgata gatggatata gaagcatttc tctgcaggat accagttcag gccccaccat 2504
tcctgccaag gccattacat cccacaaacc caaatacaaa gcagctgact tccctggatc 2564
ttccccccac tcctcacacc tcacatgtcc caggagctgc cttcattcag gcgggtagct 2624
gcactgggca tggggtggtg gtgggagctt accgccacct attcaagctc tcagctactc 2684
ctgaaacggg cagagatgat gaacagaagt gtatgtaaat acagcagcta gtgggagagc 2744
accagttggg cctaatcctg cctcatcatt cttggcagga atctgcaaat ggaaacattg 2804


CA 02488404 2007-06-05

91
tgagtatcag caatctggga agtgacaggg ttaataactc cttcccagaa gctgtatcat 2864
gagattttga ggggaccgag ccctgttaca tggatgtgaa cagtgaggat cagaggtttt 2924
atcagaacac attctttttt tctaccaact ctccagagcg tgagtatagg agtgccatga 2984
gctttttagt cagcagtttt gtaaactctg tatataaaat cattaaccac acattgtggg 3044
tgatgggaag acgatttcag ctgacagagt taatggcaac caataatggt ggcctgtagc 3104
tgctaagagc ttcacgcagg tttggcctgg gctttcactg ttggtgaatt tagagtgtcc 3164
ttttaggtgg ggcggctatt ctaaaagtgt ctttctatca ctgttaaggg ggggggaaag 3224
tgaggttcga ggatgacgta ggtaactctc ccctcccaag tccatgttcc aagtggctat 3284
gtaaagcaag atgatacaga aagctgctct aaaatctcac tgagtgattt caccttcgcc 3344
tactatgaaa tgtctcatca gacctgacat gtctgagata accaaggtga ttcaggattt 3404
gatcaaaaga agtctagtaa gaattaatta cacagaagcc tcctttcatt tctatgggcc 3464
aaacaaaggc catggataac cctacccgct ttatgtcatt acccattggg aaacacaatg 3524
gctacttctg ttagggtaca ttgaccttgg tcaagcatct taaagaaggc aaccctaatt 3584
gagagctgtc ttggctaata ctctgcacca caattgtgat gtcctagtcc taccactaga 3644
gggcatggta cagcctggca aaagttaaaa ggggtgtggc agctcccatc aggtctggag 3704
gtggtctata agcacagttg acagttgtgc attgggatgg gtggagaaag acgacaagag 3764
agcagagaat ctgctgatgt ggctgcgctt acttttagtg actttatgta cttatattaa 3824
cagctggaaa taggttgttg ggttttgagc aggctgttat agtgaggaat gttcattttt 3884
aaatgttcct aacagatttt gcttttgaaa aatgcttgtt acatgaataa tttgtggacc 3944
agggattgct tttctgaagg cagtataggg aacatgaata ttcaagatga aatacaaaaa 4004
ttatgtttaa gggtcatagt gtataagtag cttcctagga aaccctttgt gtatcttttc 4064
agactggggt gggggctgag catgcttgtg cagaaagaag ccatagccag aaaggacaga 4124
atctctcccc cactcccttg ccccataacc aaacataagc tagctagtct tgtctaatag 4184
atgggattta ctataggtga agatagccct catattcaag gacagaagct ctggcaggag 4244
taaattagca aagcagaaat agtacccttt cattcttgga ggtgctttga aattttaggt 4304
agaatataat cgaaattatg gaggttcctt agtgctcaat aatataagac ctggtgttat 4364
tagaacgagt ctttcttata aactaacaga gcaggtatat gcctgttaga ccttagctgt 4424
ggggttcctt tactattggg tgaatcatta ggtataaaaa ataatcatca accaggcaaa 4484
ttactttgct tcctagctga tgtcatccca cattggtaca ggtgttattc agtactgggt 4544
ggttcagcag ggaagccggg tgggaccagt gtgtctgtca tgaaaccact aactgcattc 4604


CA 02488404 2007-06-05

92
ctgactgaag agccatctgt catttattgg ggaaggtctt cagttgagct ctcagcctta 4664
ggaaggaagc acgtggagga gggacggagg aggttccctt gctgggcatg cttcgtagag 4724
ggccaggagc agcaggtcat gtgcacatgc cgttgcagca caagcttatg cttcccgtag 4784
ccgtggcttt tcattctgca cagtcccagg tcccagctcc cctcttatgg tttctgtcat 4844
aatgtgcttt atctgattga ctccaaacat cccgaaatgt cacctgcaga tttctcgtgg 4904
gaaccaatat gtacatgttt gcaattatgc tgtgagaatt taaatgtgtt agatggaaaa 4964
tgctattggc agggaataat aat 4987
<210> 4
<211> 414
<212> PRT
<213> Homo sapiens
<400> 4
Met Ala Ala Ala Pro Pro Ser Tyr Cys Phe Val Ala Phe Pro Pro Arg
1 5 10 15
Ala Lys Asp Gly Leu Val Val Phe Gly Lys Asn Ser Ala Arg Pro Arg
20 25 30
Asp Glu Val Gln Glu Val Val Tyr Phe Ser Ala Ala Asp His Glu Pro
35 40 45

Glu Ser Lys Val Glu Cys Thr Tyr Ile Ser Ile Asp Gln Val Pro Arg
50 55 60
Thr Tyr Ala Ile Met Ile Ser Arg Pro Ala Trp Leu Trp Gly Ala Glu
65 70 75 80
Met Gly Ala Asn Glu His Gly Val Cys Ile Ala Asn Glu Ala Ile Asn
85 90 95
Thr Arg Glu Pro Ala Ala Glu Ile Glu Ala Leu Leu Gly Met Asp Leu
100 105 110

Val Arg Leu Gly Leu Glu Arg Gly Glu Thr Ala Lys Glu Ala Leu Asp
115 120 125
Val Ile Val Ser Leu Leu Glu Glu His Gly Gln Gly Gly Asn Tyr Phe
130 135 140
Glu Asp Ala Asn Ser Cys His Ser Phe Gln Ser Ala Tyr Leu Ile Val
145 150 155 160
Asp Arg Asp Glu Ala Trp Val Leu Glu Thr Ile Gly Lys Tyr Trp Ala
165 170 175

Ala Glu Lys Val Thr Glu Gly Val Arg Cys Ile Cys Ser Gln Leu Ser
180 185 190
Leu Thr Thr Lys Met Asp Ala Glu His Pro Glu Leu Arg Ser Tyr Ala


CA 02488404 2007-06-05

93
195 200 205
Gln Ser Gln Gly Trp Trp Thr Gly Glu Gly Glu Phe Asn Phe Ser Glu
210 215 220
Val Phe Ser Pro Val Glu Asp His Leu Asp Cys Gly Ala Gly Lys Asp
225 230 235 240
Ser Leu Glu Lys Gln Glu Glu Ser Ile Thr Val Gln Thr Met Met Asn
245 250 255

Thr Leu Arg Asp Lys Ala Ser Gly Val Cys Ile Asp Ser Glu Phe Phe
260 265 270
Leu Thr Thr Ala Ser Gly Val Ser Val Leu Pro Gin Asn Arg Ser Ser
275 280 285
Pro Cys Ile His Tyr Phe Thr Gly Thr Pro Asp Pro Ser Arg Ser Ile
290 295 300

Phe Lys Pro Phe Ile Phe Val Asp Asp Val Lys Leu Val Pro Lys Thr
305 310 315 320
Gln Ser Pro Cys Phe Gly Asp Asp Asp Pro Ala Lys Lys Glu Pro Arg
325 330 335

Phe Gln Glu Lys Pro Asp Arg Arg His Glu Leu Tyr Lys Ala His Glu
340 345 350
Trp Ala Arg Ala Ile Ile Glu Ser Asp Gln Glu Gln Gly Arg Lys Leu
355 360 365
Arg Ser Thr Met Leu Glu Leu Glu Lys Gln Gly Leu Glu Ala Met Glu
370 375 380

Glu Ile Leu Thr Ser Ser Glu Pro Leu Asp Pro Ala Glu Val Gly Asp
385 390 395 400
Leu Phe Tyr Asp Cys Val Asp Thr Glu Ile Lys Phe Phe Lys
405 410
<210> 5
<211> 5345
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (489)..(2840)
<223>

<400> 5
gtacttggtt aagcagttga aacctttttt gagcaggatc tgtaaaagca taattgaatt 60
tgtttcaccc ccgtggattc cagtgggccc gacagcgcaa cagtgcctgg caacttgatg 120
catatggaag agcaatgcca agtgatctga cataatacaa attcacgaag tgacatttta 180


CA 02488404 2007-06-05

94
tcacaagcaa agttggaaat tccaaagaga agtggtgaga tctttactag tcacagtgaa 240
gatgggagaa aatgacatac ctgcagcaga tgtgggctga aaatatcctc ttctctgccc 300
aatcaggaat gctacctgtt tttgggaata aactttagag aaaggaaggg ccaaaactac 360
gacttggctt tctgaaacgg aagcataaat gttcttttcc tccatttgtc tggatctgag 420
aacctgcatt tggtattagc tagtggaagc agtatgtatg gttgaagtgc attgctgcag 480
ctggtagc atg agt ggt ggc cac cag ctg cag ctg get gcc ctc tgg ccc 530
Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro
1 5 10

tgg ctg ctg atg get acc ctg cag gca ggc ttt gga cgc aca gga ctg 578
Trp Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu
15 20 25 30
gta ctg gca gca gcg gtg gag tct gaa aga tca gca gaa cag aaa get 626
Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala
35 40 45
att atc aga gtg atc ccc ttg aaa atg gac ccc aca gga aaa ctg aat 674
Ile Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn
50 55 60
ctc act ttg gaa ggt gtg ttt get ggt gtt get gaa ata act cca gca 722
Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala
65 70 75

gaa gga aaa tta atg cag tcc cac ccg ctg tac ctg tgc aat gcc agt 770
Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala Ser
80 85 90

gat gac gac aat ctg gag cct gga ttc atc agc atc gtc aag ctg gag 818
Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys Leu Glu
95 100 105 110
agt cct cga cgg gcc ccc cgc ccc tgc ctg tca ctg get agc aag get 866
Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu Ser Leu Ala Ser Lys Ala
115 120 125
cgg atg gcg ggt gag cga gga gcc agt get gtc ctc ttt gac atc act 914
Arg Met Ala Gly Glu Arg Gly Ala Ser Ala Val Leu Phe Asp Ile Thr
130 135 140
gag gat cga get get get gag cag ctg cag cag ccg ctg ggg ctg acc 962
Glu Asp Arg Ala Ala Ala Glu Gln Leu Gln Gln Pro Leu Gly Leu Thr
145 150 155

tgg cca gtg gtg ttg atc tgg ggt aat gac get gag aag ctg atg gag 1010
Trp Pro Val Val Leu Ile Trp Gly Asn Asp Ala Glu Lys Leu Met Glu
160 165 170

ttt gtg tac aag aac caa aag gcc cat gtg agg att gag ctg aag gag 1058
Phe Val Tyr Lys Asn Gln Lys Ala His Val Arg Ile Glu Leu Lys Glu
175 180 185 190


CA 02488404 2007-06-05

ccc ccg gcc tgg cca gat tat gat gtg tgg atc cta atg aca gtg gtg 1106
Pro Pro Ala Trp Pro Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val
195 200 205
ggc acc atc ttt gtg atc atc ctg get tcg gtg ctg cgc atc cgg tgc 1154
Gly Thr Ile Phe Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys
210 215 220
cgc ccc cgc cac agc agg ccg gat ccg ctt cag cag aga aca gcc tgg 1202
Arg Pro Arg His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp
225 230 235

gcc atc agc cag ctg gcc acc agg agg tac cag gcc agc tgc agg cag 1250
Ala Ile Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln
240 245 250

gcc cgg ggt gag tgg cca gac tca ggg agc agc tgc agc tca gcc cct 1298
Ala Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro
255 260 265 270
.gtg tgt gcc atc tgt ctg gag gag ttc tct gag ggg cag gag cta cgg 1346
Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu Arg
275 280 285
gtc att tcc tgc ctc cat gag ttc cat cgt aac tgt gtg gac ccc tgg 1394
Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp Pro Trp
290 295 300
tta cat cag cat cgg act tgc ccc ctc tgc gtg ttc aac atc aca gag 1442
Leu His Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn Ile Thr Glu
305 310 315

gga gat tca ttt tcc cag tcc ctg gga ccc tct cga tct tac caa gaa 1490
Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg Ser Tyr Gln Glu
320 325 330

cca ggt cga aga ctc cac ctc att cgc cag cat ccc ggc cat gcc cac 1538
Pro Gly Arg Arg Leu His Leu Ile Arg Gln His Pro Gly His Ala His
335 340 345 350
tac cac ctc cct get gcc tac ctg ttg ggc cct tcc cgg agt gca gtg 1586
Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly Pro Ser Arg Ser Ala Val
355 360 365
get cgg ccc cca cgg cct ggt ccc ttc ctg cca tcc cag gag cca ggc 1634
Ala Arg Pro Pro Arg Pro Gly Pro Phe Leu Pro Ser Gln Glu Pro Gly
370 375 380
atg ggc cct cgg cat cac cgc ttc ccc aga get gca cat ccc cgg get 1682
Met Gly Pro Arg His His Arg Phe Pro Arg Ala Ala His Pro Arg Ala
385 390 395

cca gga gag cag cag cgc ctg gca gga gcc cag cac ccc tat gca caa 1730
Pro Gly Glu Gln Gln Arg Leu Ala Gly Ala Gln His Pro Tyr Ala Gln
400 405 410

ggc tgg gga atg agc cac ctc caa tcc acc tca cag cac cct get get 1778
Gly Trp Gly Met Ser His Leu Gln Ser Thr Ser Gln His Pro Ala Ala


CA 02488404 2007-06-05

96
415 420 425 430
tgc cca gtg ccc cta cgc cgg gcc agg ccc cct gac agc agt gga tct 1826
Cys Pro Val Pro Leu Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser
435 440 445
gga gaa agc tat tgc aca gaa cgc agt ggg tac ctg gca gat ggg cca 1874
Gly Glu Ser Tyr Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro
450 455 460
gcc agt gac tcc agc tca ggg ccc tgt cat ggc tct tcc agt gac tct 1922
Ala Ser Asp Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser
465 470 475

gtg gtc aac tgc acg gac atc agc cta cag ggg gtc cat ggc agc agt 1970
Val Val Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser
480 485 490

tct act ttc tgc agc tcc cta agc agt gac ttt gac ccc cta gtg tac 2018
Ser Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr
495 500 505 510
tgc agc cct aaa ggg gat ccc cag cga gtg gac atg cag cct agt gtg 2066
Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser Val
515 520 525
acc tct cgg cct cgt tcc ttg gac tcg gtg gtg ccc aca ggg gaa acc 2114
Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly Glu Thr
530 535 540
cag gtt tcc agc cat gtc cac tac cac cgc cac cgg cac cac cac tac 2162
Gln Val Ser Ser His Val His Tyr His Arg His Arg His His His Tyr
545 550 555

aaa aag cgg ttc cag tgg cat ggc agg aag cct ggc cca gaa acc gga 2210
Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly Pro Glu Thr Gly
560 565 570

gtc ccc cag tcc agg cct cct att cct cgg aca cag ccc cag cca gag 2258
Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr Gln Pro Gln Pro Glu
575 580 585 590
cca cct tct cct gat cag caa gtc acc gga tcc aac tca gca gcc cct 2306
Pro Pro Ser Pro Asp Gln Gln Val Thr Gly Ser Asn Ser Ala Ala Pro
595 600 605
tcg ggg cgg ctc tct aac cca cag tgc ccc agg gcc ctc cct gag cca 2354
Ser Gly Arg Leu Ser Asn Pro Gln Cys Pro Arg Ala Leu Pro Glu Pro
610 615 620
gcc cct ggc cca gtt gac gcc tcc agc atc tgc ccc agt acc agc agt 2402
Ala Pro Gly Pro Val Asp Ala Ser Ser Ile Cys Pro Ser Thr Ser Ser
625 630 635

ctg ttc aac ttg caa aaa tcc agc ctc tct gcc cga cac cca cag agg 2450
Leu Phe Asn Leu Gln Lys Ser Ser Leu Ser Ala Arg His Pro Gln Arg
640 645 650


CA 02488404 2007-06-05

97
aaa agg cgg ggg ggt ccc tcc gag ccc acc cct ggc tct cgg ccc cag 2498
Lys Arg Arg Gly Gly Pro Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln
655 660 665 670
gat gca act gtg cac cca get tgc cag att ttt ccc cat tac acc ccc 2546
Asp Ala Thr Val His Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro
675 680 685
agt gtg gca tat cct tgg tcc cca gag gca cac ccc ttg atc tgt gga 2594
Ser Val Ala Tyr Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly
690 695 700
cct cca ggc ctg gac aag agg ctg cta cca gaa acc cca ggc ccc tgt 2642
Pro Pro Gly Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys
705 710 715

tac tca aat tca cag cca gtg tgg ttg tgc ctg act cct cgc cag ccc 2690
Tyr Ser Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro
720 725 730

ctg gaa cca cat cca cct ggg gag ggg cct tct gaa tgg agt tct gac 2738
Leu Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp
735 740 745 750
acc gca gag ggc agg cca tgc cct tat ccg cac tgc cag gtg ctg tcg 2786
Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu Ser
755 760 765
gcc cag cct ggc tca gag gag gaa ctc gag gag ctg tgt gaa cag get 2834
Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala
770 775 780
gtg tga gatgttcagg cctagctcca accaagagtg tgctccagat gtgtttgggc 2890
Val

cctacctggc acagagtcct gctcctggga aaggaaagga ccacagcaaa caccattctt 2950
tttgccgtac ttcctagaag cactggaaga ggactggtga tggtggaggg tgagagggtg 3010
ccgtttcctg ctccagctcc agaccttgtc tgcagaaaac atctgcagtg cagcaaatcc 3070
atgtccagcc aggcaaccag ctgctgcctg tggcgtgtgt gggctggatc ccttgaaggc 3130
tgagtttttg agggcagaaa gctagctatg ggtagccagg tgttacaaag gtgctgctcc 3190
ttctccaacc cctacttggt ttccctcacc ccaagcctca tgttcatacc agccagtggg 3250
ttcagcagaa cgcatgacac cttatcacct ccctccttgg gtgagctctg aacaccagct 3310
ttggcccctc cacagtaagg ctgctacatc aggggcaacc ctggctctat cattttcctt 3370
ttttgccaaa aggaccagta gcataggtga gccctgagca ctaaaaggag gggtccctga 3430
agctttccca ctatagtgtg gagttctgtc cctgaggtgg gtacagcagc cttggttcct 3490
ctgggggttg agaataagaa tagtggggag ggaaaaactc ctccttgaag atttcctgtc 3550
tcagagtccc agagaggtag aaaggaggaa tttctgctgg actttatctg ggcagaggaa 3610


CA 02488404 2007-06-05

98
ggatggaatg aaggtagaaa aggcagaatt acagctgagc ggggacaaca aagagttctt 3670
ctctgggaaa agttttgtct tagagcaagg atggaaaatg gggacaacaa aggaaaagca 3730
aagtgtgacc cttgggtttg gacagcccag aggcccagct ccccagtata agccatacag 3790
gccagggacc cacaggagag tggattagag cacaagtctg gcctcactga gtggacaaga 3850
gctgatgggc ctcatcaggg tgacattcac cccagggcag cctgaccact cttggcccct 3910
caggcattat cccatttgga atgtgaatgt ggtggcaaag tgggcagagg accccacctg 3970
ggaacctttt tccctcagtt agtggggaga ctagcaccta ggtacccaca tgggtattta 4030
tatctgaacc agacagacgc ttgaatcagg cactatgtta agaaatatat ttatttgcta 4090
atatatttat ccacaaacag gcactatgtt aagaaatata tttatttgct aatatattta 4150
tccacaaatg tggtctggtc ttgtggtttt gttctgtcgt gactgtcact cagggtaaca 4210
acgtcatctc tttctacatc aagagaagta aattatttat gttatcagag gctaggctcc 4270
gattcatgaa aggatagggt agagtagagg gcttggcaat aagaactggt ttgtaagccc 4330
ctaaaagtgt ggcttagtga gatcagggaa ggagaaagca tgactggatt cttactgtgc 4390
ttcagtcatt attattatac tgttcacttc acacattatc atacttcagt gactcagacc 4450
ttgggcaaat actctgtgcc tcgctttttc agtccataaa atgggcctac ttaatagttg 4510
ttgcaggact tacatgagat aatagagtgt agaaaatatg ttccaaagtg gaaagtttta 4570
ttcatgtgat agaaaacatc caaacctgtc acagagccca tctgaacaca gcatgggacc 4630
gccaacaaga agaaagcccg cccggaagca gctcaatcag gaggctgggc tggaatgaca 4690
gcgcagcggg gcctgaaact atttatatcc caaagctcct ctcagataaa cacaaatgac 4750
tgcgttctgc ctgcactcgg gctattgcga ggacagagag ctggtgctcc attggcgtga 4810
agtctccagg gccagaaggg gcctttgtcg cttcctcaca aggcacaagt tccccttctg 4870
cttccccgag aaaggtttgg taggggtggt ggtttagtgc ctatagaaca aggcatttcg 4930
cttcctagac ggtgaaatga aagggaaaaa aaggacacct aatctcctac aaatggtctt 4990
tagtaaagga accgtgtcta agcgctaaga actgcgcaaa gtataaatta tcagccggaa 5050
cgagcaaaca gacggagttt taaaagataa atacgcattt ttttccgccg tagctcccag 5110
gccagcattc ctgtgggaag caagtggaaa ccctatagcg ctctcgcagt taggaaggag 5170
gggtggggct gtcgctggat ttcttctcgg tctctgcaga gacaatccag agggagacag 5230
tggattcact gcccccaatg cttctaaaac ggggagacaa aacaaaaaaa aacaaacttc 5290
cgggttacca tcggggaaca ggaccgacgc ccagggccac cagccccctc gtgcc 5345


CA 02488404 2007-06-05

99
<210> 6
<211> 783
<212> PRT
<213> Homo sapiens
<400> 6
Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu
1 5 10 15
Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu Val Leu
20 25 30
Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys Ala Ile Ile
35 40 45

Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys Leu Asn Leu Thr
50 55 60
Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile Thr Pro Ala Glu Gly
65 70 75 80
Lys Leu Met Gln Ser His Pro Leu Tyr Leu Cys Asn Ala Ser Asp Asp
85 90 95
Asp Asn Leu Glu Pro Gly Phe Ile Ser Ile Val Lys Leu Glu Ser Pro
100 105 110

Arg Arg Ala Pro Arg Pro Cys Leu Ser Leu Ala Ser Lys Ala Arg Met
115 120 125
Ala Gly Glu Arg Gly Ala Ser Ala Val Leu Phe Asp Ile Thr Glu Asp
130 135 140
Arg Ala Ala Ala Glu Gln Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro
145 150 155 160
Val Val Leu Ile Trp Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val
165 170 175

Tyr Lys Asn Gln Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro
180 185 190
Ala Trp Pro Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr
195 200 205
Ile Phe Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro
210 215 220

Arg His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile
225 230 235 240
Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala Arg
245 250 255

Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro Val Cys
260 265 270
Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu Arg Val Ile


CA 02488404 2007-06-05

100
275 280 285
Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp Pro Trp Leu His
290 295 300
Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn Ile Thr Glu Gly Asp
305 310 315 320
Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg Ser Tyr Gln Glu Pro Gly
325 330 335

Arg Arg Leu His Leu Ile Arg Gln His Pro Gly His Ala His Tyr His
340 345 350
Leu Pro Ala Ala Tyr Leu Leu Gly Pro Ser Arg Ser Ala Val Ala Arg
355 360 365
Pro Pro Arg Pro Gly Pro Phe Leu Pro Ser Gln Glu Pro Gly Met Gly
370 375 380

Pro Arg His His Arg Phe Pro Arg Ala Ala His Pro Arg Ala Pro Gly
385 390 395 400
Glu Gln Gln Arg Leu Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp
405 410 415

Gly Met Ser His Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro
420 425 430
Val Pro Leu Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu
435 440 445
Ser Tyr Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser
450 455 460

Asp Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val
465 470 475 480
Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser Thr
485 490 495

Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr Cys Ser
500 505 510
Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser Val Thr Ser
515 520 525
Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly Glu Thr Gln Val
530 535 540

Ser Ser His Val His Tyr His Arg His Arg His His His Tyr Lys Lys
545 550 555 560
Arg Phe Gln Trp His Gly Arg Lys Pro Gly Pro Glu Thr Gly Val Pro
565 570 575

Gln Ser Arg Pro Pro Ile Pro Arg Thr Gln Pro Gln Pro Glu Pro Pro
580 585 590


CA 02488404 2007-06-05

101
Ser Pro Asp Gln Gln Val Thr Gly Ser Asn Ser Ala Ala Pro Ser Gly
595 600 605

Arg Leu Ser Asn Pro Gln Cys Pro Arg Ala Leu Pro Glu Pro Ala Pro
610 615 620
Gly Pro Val Asp Ala Ser Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe
625 630 635 640
Asn Leu Gln Lys Ser Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg
645 650 655
Arg Gly Gly Pro Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala
660 665 670

Thr Val His Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val
675 680 685
Ala Tyr Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro
690 695 700
Gly Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser
705 710 715 720
Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu Glu
725 730 735

Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp Thr Ala
740 745 750
Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu Ser Ala Gln
755 760 765
Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu Gln Ala Val
770 775 780
<210> 7
<211> 22
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 7
acaacagcct caagatcatc ag 22
<210> 8
<211> 20
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 8
ggtccaccac tgacacgttg 20


CA 02488404 2007-06-05

102
<210> 9
<211> 22
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 9
agctgagaca tttgttctct tg 22
<210> 10
<211> 22
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 10
tataaaccag ctgagtccag ag 22
<210> 11
<211> 23
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 11
ttcccgatat caacatctac cag 23
<210> 12
<211> 23
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 12
agtgtgtgac ctcaataagg cat 23
<210> 13
<211> 27
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 13


CA 02488404 2007-06-05

103
caggctttgg acgcacagga ctggtac 27
<210> 14
<211> 27
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 14
ctttgtgatc atcctggctt cggtgct 27
<210> 15
<211> 22
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 15
ggttgagatt taagttctca as 22
<210> 16
<211> 41
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 16
agttaagctt gccgggatga cttctcagcg ttcccctctg g 41
<210> 17
<211> 32
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 17
atctcgagta ccaaggaccc ggcccgactc tg 32
<210> 18
<211> 34
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence


CA 02488404 2007-06-05

104
<400> 18
gcggatccag gatggctgct gcagctcctc caag 34
<210> 19
<211> 36
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 19
tagaattctt aaagaactta atctccgtgt caacac 36
<210> 20
<211> 31
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 20
tgcagatctg cagctggtag catgagtggt g 31
<210> 21
<211> 32
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 21
gaggagctgt gtgaacaggc tgtgtgagat gt 32
<210> 22
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 22
tctgcacggt gagtag 16
<210> 23
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide


CA 02488404 2007-06-05

105
<400> 23
ctactcaccg tgcaga 16
<210> 24
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 24
ttctgtaggt gttgca 16
<210> 25
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 25
tgcaacacct acagaa 16
<210> 26
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 26
cttttcagga tggctg 16
<210> 27
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 27
cagccatcct gaaaag 16
<210> 28
<211> 16
<212> DNA
<213> Artificial

<220>


CA 02488404 2007-06-05

106
<223> an artificially synthesized S-oligonucleotide
<400> 28
aggttgaggt aagccg 16
<210> 29
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 29
cggcttacct caacct 16
<210> 30
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 30
tggtagcatg agtggt 16
<210> 31
<211> 16
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized S-oligonucleotide
<400> 31
accactcatg ctacca 16
<210> 32
<211> 22
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 32
tggtagccaa gtgcaggtta to 22
<210> 33
<211> 22
<212> DNA
<213> Artificial


CA 02488404 2007-06-05

107
<220>
<223> an artificially synthesized primer sequence
<400> 33
ccaaagggtt tctgcagttt ca 22
<210> 34
<211> 30
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 34
tgcggatcca gagcagattg tactgagagt 30
<210> 35
<211> 29
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 35
ctctatctcg agtgaggcgg aaagaacca 29
<210> 36
<211> 47
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 36
tttaagcttg aagaccattt ttggaaaaaa aaaaaaaaaa aaaaaac 47
<210> 37
<211> 34
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 37
tttaagcttg aagacatggg aaagagtggt ctca 34
<210> 38
<211> 51
<212> DNA
<213> Artificial


CA 02488404 2007-06-05

108
<220>
<223> an artificially synthesized oligonucleotide sequence
<400> 38
caccgaagca gcacgacttc ttcttcaaga gagaagaagt cgtgctgctt c 51
<210> 39
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide sequence
<400> 39
aaaagaagca gcacgacttc ttctctcttg aagaagaagt cgtgctgctt c 51
<210> 40
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 40
tcccgtcacc ggatccaact cagttcaaga gactgagttg gatccggtga c 51
<210> 41
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 41
aaaagtcacc ggatccaact cagtctcttg aactgagttg gatccggtga c 51
<210> 42
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 42
tcccgctatt gcacagaacg cagttcaaga gactgcgttc tgtgcaatag c 51
<210> 43
<211> 51
<212> DNA


CA 02488404 2007-06-05

109
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 43
aaaagctatt gcacagaacg cagtctcttg aactgcgttc tgtgcaatag c 51
<210> 44
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 44
tccccagaaa gctgttatca gagttcaaga gactctgata acagctttct g 51
<210> 45
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 45
aaaacagaaa gctgttatca gagtctcttg aactctgata acagctttct g 51
<210> 46
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 46
tccctgagcc acctccaatc cacttcaaga gagtggattg gaggtggctc a 51
<210> 47
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 47
aaaatgagcc acctccaatc cactctcttg aagtggattg gaggtggctc a 51
<210> 48
<211> 51


CA 02488404 2007-06-05

110
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 48
tcccctgcac ggacatcagc ctattcaaga gataggctga tgtccgtgca g 51
<210> 49
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 49
aaaactgcac ggacatcagc ctatctcttg aataggctga tgtccgtgca g 51
<210> 50
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 50
tcccgtgtca gagagccctg ggattcaaga gatcccaggg ctctctgaca c 51
<210> 51
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 51
aaaagtgtca gagagccctg ggatctcttg aatcccaggg ctctctgaca c 51
<210> 52
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 52
tccccctcaa tgtcatttgg atgttcaaga gacatccaaa tgcaattgag g 51
<210> 53


CA 02488404 2007-06-05

111
<211> 51
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 53
aaaacctcaa tgtcatttgg atgtctcttg aacatccaaa tgcaattgag g 51
<210> 54
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 54
tccctgtcat ttggatggtc actttcaaga gaagtgacca tccaaatgac a 51
<210> 55
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 55
aaaatgtcat ttggatggtc acttctcttg aaagtgacca tccaaatgac a 51
<210> 56
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 56
tccctgccaa ccaacctgaa cagttcaaga gactgttcag gttggttggc a 51
<210> 57
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 57
aaaatgccaa ccaacctgaa cagtctcttg aactgttcag gttggttggc a 51


CA 02488404 2007-06-05

112
<210> 58
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for SiRNA
<400> 58
tcccccaacc tgaacaggtc atcttcaaga gagatgacct gttcaggttg g 51
<210> 59
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 59
aaaaccaacc tgaacaggtc atctctcttg aagatgacct gttcaggttg g 51
<210> 60
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 60
tccccctgaa caggtcatcc tgtttcaaga gaacaggatg acctgttcag g 51
<210> 61
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 61
aaaacctgaa caggtcatcc tgttctcttg aaacaggatg acctgttcag g 51
<210> 62
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 62
tccccaggtc atcctgtatc aggttcaaga gacctgatac aggatgacct g 51


CA 02488404 2007-06-05

113
<210> 63
<211> 51
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized oligonucleotide for siRNA
<400> 63
aaaacaggtc atcctgtatc aggtctcttg aacctgatac aggatgacct g 51
<210> 64
<211> 28
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 64
gaagatctgc agcggtggag tctgaaag 28
<210> 65
<211> 31
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 65
ggaattcgga ctgggaaaat gaatctccct c 31
<210> 66
<211> 26
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 66
ggagatctcc tgatcagcaa gtcacc 26
<210> 67
<211> 31
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 67
ggaattccac agcctgttca cacagctcct c 31


'CA 02488404 2007-06-05

114
<210> 68
<211> 31
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 68
gcggatccag gatggctgca gctcctccaa g 31
<210> 69
<211> 38
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized primer sequence
<400> 69
ctgaattcac ttaaagaact taatctccgt gtcaacac 38
<210> 70
<211> 10
<212> DNA
<213> Homo sapiens
<400> 70
cccgggatga 10
<210> 71
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 71

Ser Tyr Gln Glu Pro Gly Arg Arg Leu
1 5

<210> 72
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 72


CA 02488404 2007-06-05

115
His Tyr His Leu Pro Ala Ala Tyr Leu
1 5
<210> 73
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 73

Leu Phe Asn Leu Gln Lys Ser Ser Leu
1 5

<210> 74
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 74

Gly Phe Gly Arg Thr Gly Leu Val Leu
1 5

<210> 75
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 75

Arg Tyr Gln Ala Ser Cys Arg Gln Ala
1 5

<210> 76
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 76

Arg Ala Pro Gly Glu Gln Gln Arg Leu
1 5


CA 02488404 2007-06-05

116
<210> 77
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 77

Arg Ala Pro Arg Pro Cys Leu Ser Leu
1 5

<210> 78
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 78

Arg Pro Pro Arg Pro Gly Pro Phe Leu
1 5

<210> 79
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 79

Lys Ala Val Ile Arg Val Ile Pro Leu
1 5

<210> 80
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 80

Asn Ser Gln Pro Val Trp Leu Cys Leu
1 5

<210> 81
<211> 10


CA 02488404 2007-06-05

117
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 81

Ser Tyr Cys Thr Glu Arg Ser Gly Tyr Leu
1 5 10
<210> 82
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 82

His Tyr His Leu Pro Ala Ala Tyr Leu Leu
1 5 10
<210> 83
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 83

Cys Tyr Ser Asn Ser Gln Pro Val Trp Leu
1 5 10
<210> 84
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 84

Ile Phe Val Ile Ile Leu Ala Ser Val Leu
1 5 10
<210> 85
<211> 10
<212> PRT
<213> Artificial


CA 02488404 2007-06-05

118
<220>
<223> an artificially synthesized peptide sequence
<400> 85

Val Phe Asn Ile Thr Glu Gly Asp Ser Phe
1 5 10
<210> 86
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 86

Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe
1 5 10
<210> 87
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 87

Lys Leu Met Gln Ser His Pro Leu Tyr Leu
1 5 10
<210> 88
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 88

Lys Met Asp Pro Thr Gly Lys Leu Asn Leu
1 5 10
<210> 89
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence


CA 02488404 2007-06-05

119
<400> 89

His Tyr Thr Pro Ser Val Ala Tyr Pro Trp
1 5 10
<210> 90
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 90

Gly Gln Glu Leu Arg Val Ile Ser Cys Leu
1 5 10
<210> 91
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 91

Asn Phe Gln Pro Val Trp Leu Cys Leu
1 5

<210> 92
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 92

Asn Tyr Gln Pro Val Trp Leu Cys Leu
1 5

<210> 93
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 93


CA 02488404 2007-06-05

120
Lys Leu Asn Leu Thr Leu Glu Gly Val
1 5
<210> 94
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 94

Gln Leu Ala Ala Leu Trp Pro Trp Leu
1 5

<210> 95
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 95

Leu Met Gln Ser His Pro Leu Tyr Leu
1 5

<210> 96
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 96

Leu Leu Gly Pro Ser Arg Ser Ala Val
1 5

<210> 97
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 97

Ala Leu Trp Pro Trp Leu Leu Met Ala
1 5


CA 02488404 2007-06-05

121
<210> 98
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 98

Trp Leu Leu Met Ala Thr Leu Gln Ala
1 5

<210> 99
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 99

Trp Ile Leu Met Thr Val Val Gly Thr
1 5

<210> 100
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 100

Lys Leu Met Glu Phe Val Tyr Lys Asn
1 5

<210> 101
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 101

Asn Leu Thr Leu Glu Gly Val Phe Ala
1 5

<210> 102
<211> 9


CA 02488404 2007-06-05

122
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 102

Gly Leu Thr Trp Pro Val Val Leu Ile
1 5

<210> 103
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 103

Tyr Leu Leu Gly Pro Ser Arg Ser Ala Val
1 5 10
<210> 104
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 104

Leu Met Thr Val Val Gly Thr Ile Phe Val
1 5 10
<210> 105
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 105

Cys Leu His Glu Phe His Arg Asn Cys Val
1 5 10
<210> 106
<211> 10
<212> PRT
<213> Artificial


CA 02488404 2007-06-05

123
<220>
<223> an artificially synthesized peptide sequence
<400> 106

Ser Leu Ser Ser Asp Phe Asp Pro Leu Val
1 5 10
<210> 107
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 107

Gln Leu Ala Ala Ile Trp Pro Trp Leu Leu
1 5 10
<210> 108
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 108

Ala Leu Trp Pro Trp Leu Leu Met Ala Thr
1 5 10
<210> 109
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 109

Leu Gln Leu Ala Ala Leu Trp Pro Trp Leu
1 5 10
<210> 110
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence


CA 02488404 2007-06-05

124
<400> 110

Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu
1 5 10
<210> 111
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 111

Trp Leu His Gin His Arg Thr Cys Pro Leu
1 5 10
<210> 112
<211> 20
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized target sequence for s.iRNA
<400> 112
gtcaccggat ccaactcagt 20
<210> 113
<211> 20
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized target sequence for s.iRNA
<400> 113
gctattgcac agaacgcagt 20
<210> 114
<211> 20
<212> DNA
<213> Artificial

<220>
<223> an artificially synthesized target sequence for s:LRNA
<400> 114
caggtcatcc tgtatcaggt 20
<210> 115
<211> 9


CA 02488404 2007-06-05

125
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 115
Tyr Leu Trp Glu Lys Leu Asp Asn Thr
1 5

<210> 116
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 116
Leu Leu Leu Leu Ser Leu His Gly Val
1 5

<210> 117
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 117
Ile Asn Leu Asn Val Ile Trp Met Val
1 5

<210> 118
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 118
Trp Met Val Thr Pro Leu Ser Asn Ala
1 5

<210> 119
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 119


CA 02488404 2007-06-05

126
Cys Leu Val Asn Asn Leu Pro Asp Ile
1 5
<210> 120
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 120
Ser Leu His Gly Val Ala Ala Ser Leu
1 5

<210> 121
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 121
Val Ile Ile Ile Phe Cys Ile Ala Leu
1 5

<210> 122
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 122
Leu Ile Asn Leu Asn Val Ile Trp Met
1 5

<210> 123
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 123
Ala Val Leu Pro Cys Thr Phe Thr Thr
1 5

<210> 124
<211> 9
<212> PRT


CA 02488404 2007-06-05

127
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 124
Ala Leu Ser Ser Gly Leu Tyr Gln Cys
1 5

<210> 125
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 125
Val Met Ser Arg Ser Asn Gly Ser Val
1 5

<210> 126
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 126
Ser Ile Phe Ile Asn Asn Thr Gln Leu
1 5

<210> 127
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 127
Lys Val His Arg Asn Thr Asp Ser Val
1 5

<210> 128
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 128
Arg Ile Gly Ala Val Pro Val Met Val


CA 02488404 2007-06-05

128
1 5

<210> 129
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 129
Asn Ile Gly Val Thr Gly Leu Thr Val
1 5

<210> 130
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 130
Ser Ile Tyr Ala Asn Gly Thr His Leu
1 5

<210> 131
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 131
Leu Leu Cys Ser Ser Glu Glu Gly Ile
1 5

<210> 132
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 132
Leu Leu Ser Leu His Gly Val Ala Ala
1 5

<210> 133
<211> 9
<212> PRT
<213> Artificial


CA 02488404 2007-06-05

129
<220>
<223> an artificially synthesized peptide sequence
<400> 133
Ile Ile Phe Cys Ile Ala Leu Ile Leu
1 5

<210> 134
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 134
Thr Met Pro Ala Thr Asn Val Ser Ile
1 5

<210> 135
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 135
Tyr Leu Trp Glu Lys Leu Asp Asn Thr Leu
1 5 10
<210> 136
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 136
Leu Ile Asn Leu Asn Val Ile Trp Met Val
1 5 10
<210> 137
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 137
Ala Leu Ser Ser Gly Leu Tyr Gln Cys Val
1 5 10


CA 02488404 2007-06-05

130
<210> 138
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 138
Ala Leu Ile Asn Leu Asn Val Ile Trp Met
1 5 10
<210> 139
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 139
Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile
1 5 10
<210> 140
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 140
Val Leu Pro Cys Thr Phe Thr Thr Ser Ala
1 5 10
<210> 141
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 141
Leu Leu Leu Ser Leu His Gly Val Ala Ala
1 5 10
<210> 142
<211> 10
<212> PRT
<213> Artificial


CA 02488404 2007-06-05

131
<220>
<223> an artificially synthesized peptide sequence
<400> 142
Ser Ile Tyr Ala Asn Gly Thr His Leu Val
1 5 10
<210> 143
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 143
Gln Leu Ser Asp Thr Gly Thr Tyr Gln Cys
1 5 10
<210> 144
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 144
Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala
1 5 10
<210> 145
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 145
Pro Leu Leu Leu Leu Ser Leu His Gly Val
1 5 10
<210> 146
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 146
Ile Gln Val Ala Arg Gly Gln Pro Ala Val
1 5 10


CA 02488404 2007-06-05

132
<210> 147
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 147
Phe Ile Asn Asn Thr Gln Leu Ser Asp Thr
1 5 10
<210> 148
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 148
Leu Val Pro Gly Gln His Lys Thr Leu Val
1 5 10
<210> 149
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 149
Asn Leu Pro Asp Ile Gly Gly Arg Asn Ile
1 5 10
<210> 150
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 150
Val Leu Val Pro Pro Ser Ala Pro His Cys
1 5 10
<210> 151
<211> 10
<212> PRT
<213> Artificial

<220>


CA 02488404 2007-06-05

133
<223> an artificially synthesized peptide sequence
<400> 151
Ala Val Ile Ile Ile Phe Cys Ile Ala Leu
1 5 10
<210> 152
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 152
Val Ile Ile Ile Phe Cys Ile Ala Leu Ile
1 5 10
<210> 153
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 153
Ile Leu Gly Ala Phe Phe Tyr Trp Arg Ser
1 5 10
<210> 154
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 154
Gly Leu Thr Val Leu Val Pro Pro Ser Ala
1 5 10
<210> 155
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 155
Ser Ile Phe Lys Pro Phe Ile Phe Val
1 5


CA 02488404 2007-06-05

134
<210> 156
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 156
Trp Leu Trp Gly Ala Glu Met Gly Ala
1 5

<210> 157
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 157
Ile Met Ile Ser Arg Pro Ala Trp Leu
1 5

<210> 158
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 158
Leu Leu Gly Met Asp Leu Val Arg Leu
1 5

<210> 159
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 159
Phe Ile Phe Val Asp Asp Val Lys Leu
1 5

<210> 160
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence


CA 02488404 2007-06-05

135
<400> 160
Val Cys Ile Asp Ser Glu Phe Phe Leu
1 5

<210> 161
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 161
Lys Pro Phe Ile Phe Val Asp Asp Val
1 5

<210> 162
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 162
Ile Val Asp Arg Asp Glu Ala Trp Val
1 5

<210> 163
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 163
Thr Leu Arg Asp Lys Ala Ser Gly Val
1 5

<210> 164
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 164
Lys Met Asp Ala Glu His Pro Glu Leu
1 5

<210> 165


CA 02488404 2007-06-05

136
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 165
Ala Leu Asp Val Ile Val Ser Leu Leu
1 5

<210> 166
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 166
Tyr Ala Gln Ser Gln Gly Trp Trp Thr
1 5

<210> 167
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 167
Lys Leu Arg Ser Thr Met Leu Glu Leu
1 5

<210> 168
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 168
Tyr Leu Ile Val Asp Arg Asp Glu Ala
1 5

<210> 169
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence


CA 02488404 2007-06-05

137
<400> 169
Ala Ala Pro Pro Ser Tyr Cys Phe Val
1 5

<210> 170
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 170
Gly Met Asp Leu Val Arg Leu Gly Leu
1 5

<210> 171
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 171
Lys Val Thr Glu Gly Val Arg Cys Ile
1 5

<210> 172
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 172
Cys Ile Asp Ser Glu Phe Phe Leu Thr
1 5

<210> 173
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 173
Thr Val Gln Thr Met Met Asn Thr Leu
1 5

<210> 174


CA 02488404 2007-06-05

138
<211> 9
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 174
Glu Met Gly Ala Asn Glu His Gly Val
1 5

<210> 175
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 175
Phe Ile Phe Val Asp Asp Val Lys Leu Val
1 5 10
<210> 176
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 176
Leu Ile Val Asp Arg Asp Glu Ala Trp Val
1 5 10
<210> 177
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 177
Phe Leu Thr Thr Ala Ser Gly Val Ser Val
1 5 10
<210> 178
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence


CA 02488404 2007-06-05

139
<400> 178
Thr Met Leu Glu Leu Glu Lys Gln Gly Leu
1 5 10
<210> 179
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 179
Ala Leu Leu Gly Met Asp Leu Val Arg Leu
1 5 10
<210> 180
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 180
Ala Ile Met Ile Ser Arg Pro Ala Trp Leu
1 5 10
<210> 181
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 181
Gly Val Cys Ile Asp Ser Glu Phe Phe Leu
1 5 10
<210> 182
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 182
Lys Leu Val Pro Lys Thr Gln Ser Pro Cys
1 5 10
<210> 183
<211> 10


CA 02488404 2007-06-05

140
<212> PRT
<213> Artificial
<220>
<223> an artificially synthesized peptide sequence
<400> 183

Phe Asn Phe Ser Glu Val Phe Ser Pro Val
1 5 10
<210> 184
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 184
Tyr Ile Ser Ile Asp Gln Val Pro Arg Thr
1 5 10
<210> 185
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 185
Gly Glu Gly Glu Phe Asn Phe Ser Glu Val
1 5 10
<210> 186
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 186
Trp Ala Ala Glu Lys Val Thr Glu Gly Val
1 5 10
<210> 187
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 187


CA 02488404 2007-06-05

141
Val Leu Pro Gln Asn Arg Ser Ser Pro Cys
1 5 10
<210> 188
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 188
Ala Ala Ala Pro Pro Ser Tyr Cys Phe Val
1 5 10
<210> 189
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 189
Thr Met Met Asn Thr Leu Arg Asp Lys Ala
1 5 10
<210> 190
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 190
Glu Val Gly Asp Leu Phe Tyr Asp Cys Val
1 5 10
<210> 191
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 191
Ala Glu Met Gly Ala Asn Glu His Gly Val
1 5 10
<210> 192
<211> 10
<212> PRT


CA 02488404 2007-06-05

142
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 192
Gly Leu Val Val Phe Gly Lys Asn Ser Ala
1 5 10
<210> 193
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 193
Gln Leu Ser Leu Thr Thr Lys Met Asp Ala
1 5 10
<210> 194
<211> 10
<212> PRT
<213> Artificial

<220>
<223> an artificially synthesized peptide sequence
<400> 194
Arg Ser Ile Phe Lys Pro Phe Ile Phe Val
1 5 10

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date 2012-11-27
(86) PCT Filing Date 2003-06-03
(87) PCT Publication Date 2003-12-18
(85) National Entry 2004-12-03
Examination Requested 2008-06-02
(45) Issued 2012-11-27
Expired 2023-06-05

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $400.00 2004-12-03
Maintenance Fee - Application - New Act 2 2005-06-03 $100.00 2004-12-03
Registration of a document - section 124 $100.00 2005-02-22
Registration of a document - section 124 $100.00 2005-02-22
Maintenance Fee - Application - New Act 3 2006-06-05 $100.00 2006-04-27
Registration of a document - section 124 $100.00 2006-11-17
Maintenance Fee - Application - New Act 4 2007-06-04 $100.00 2007-05-17
Maintenance Fee - Application - New Act 5 2008-06-03 $200.00 2008-05-26
Request for Examination $800.00 2008-06-02
Maintenance Fee - Application - New Act 6 2009-06-03 $200.00 2009-05-26
Maintenance Fee - Application - New Act 7 2010-06-03 $200.00 2010-05-24
Maintenance Fee - Application - New Act 8 2011-06-03 $200.00 2011-05-20
Maintenance Fee - Application - New Act 9 2012-06-04 $200.00 2012-05-22
Final Fee $846.00 2012-09-13
Maintenance Fee - Patent - New Act 10 2013-06-03 $250.00 2013-05-21
Maintenance Fee - Patent - New Act 11 2014-06-03 $250.00 2014-05-26
Maintenance Fee - Patent - New Act 12 2015-06-03 $250.00 2015-05-22
Maintenance Fee - Patent - New Act 13 2016-06-03 $250.00 2016-05-25
Maintenance Fee - Patent - New Act 14 2017-06-05 $250.00 2017-05-24
Maintenance Fee - Patent - New Act 15 2018-06-04 $450.00 2018-05-18
Maintenance Fee - Patent - New Act 16 2019-06-03 $450.00 2019-05-20
Maintenance Fee - Patent - New Act 17 2020-06-03 $450.00 2020-05-25
Maintenance Fee - Patent - New Act 18 2021-06-03 $459.00 2021-05-25
Maintenance Fee - Patent - New Act 19 2022-06-03 $458.08 2022-05-23
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
ONCOTHERAPY SCIENCE, INC.
Past Owners on Record
FURUKAWA, YOICHI
JAPAN AS REPRESENTED BY THE PRESIDENT OF THE UNIVERSITY OF TOKYO
NAKAMURA, YUSUKE
TAHARA, HIDEAKI
THE UNIVERSITY OF TOKYO
TSUNODA, TAKUYA
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2004-12-03 2 74
Claims 2004-12-03 7 417
Drawings 2004-12-03 44 1,773
Description 2004-12-03 156 6,895
Representative Drawing 2005-03-21 1 9
Cover Page 2005-03-22 1 42
Claims 2007-06-01 4 233
Description 2007-06-01 142 6,673
Description 2007-06-05 142 6,656
Claims 2007-08-30 7 408
Claims 2008-06-02 8 396
Claims 2010-12-21 8 289
Description 2010-12-21 142 6,622
Claims 2011-04-05 8 289
Claims 2011-11-30 6 203
Claims 2012-04-02 5 166
Cover Page 2012-11-01 1 45
Assignment 2006-02-06 28 749
PCT 2004-12-03 13 571
Assignment 2004-12-03 4 104
PCT 2004-12-03 1 51
Assignment 2005-02-22 21 654
Correspondence 2005-04-20 1 28
Correspondence 2005-08-22 1 22
Prosecution-Amendment 2005-08-15 1 57
Correspondence 2006-04-06 1 18
Fees 2006-04-27 1 40
Assignment 2006-05-26 5 173
Assignment 2006-11-17 2 81
Prosecution-Amendment 2007-06-01 66 1,669
Prosecution-Amendment 2007-06-05 62 1,426
Prosecution-Amendment 2007-08-30 9 449
Prosecution-Amendment 2008-06-02 18 900
Prosecution-Amendment 2008-06-02 1 37
Prosecution-Amendment 2010-07-08 5 257
Prosecution-Amendment 2010-12-21 26 1,357
Prosecution-Amendment 2011-04-05 3 87
Prosecution-Amendment 2011-06-17 3 108
Prosecution-Amendment 2011-11-30 9 338
Prosecution-Amendment 2012-02-03 2 55
Prosecution Correspondence 2009-03-02 1 32
Prosecution-Amendment 2012-04-02 7 247
Correspondence 2012-09-13 1 43

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